Simultaneous gene silencing and suppressing gene silencing in ihe same cell

ABSTRACT

The present invention relates to genetically modified cells that are capable of optimal transgene expression by co-expressing a silencing suppressor whilst at the same time are also capable of silencing a gene, such as a naturally occurring gene of the cell. The present invention also relates to methods of producing the modified cells, as well as relates to processes for obtaining a genetically modified cell with a desired property.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/369,579, filed Jun. 7, 2014, now U.S. Pat. No. 10,822,615,granted Nov. 3, 2020, a § 371 national stage of PCT InternationalApplication No. PCT/AU2012/001594, filed Dec. 21, 2012, claiming thebenefit of U.S. Provisional Application No. 61/580,574, filed Dec. 27,2011, the contents of each of which are hereby incorporated by referenceinto the application.

REFERENCE TO A SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“201007_83667-AA-PCT-US_Substitute_Sequence_Listing_LMO.txt,” which is80.9 kilobytes in size and was created Oct. 4, 2020 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Oct. 7, 2020 aspart of this application.

FIELD OF THE INVENTION

The present invention relates to genetically modified cells that arecapable of optimal transgene expression by co-expressing a silencingsuppressor whilst at the same time are also capable of silencing a gene,such as a naturally occurring gene of the cell. The present inventionalso relates to methods of producing the modified cells, as well asrelates to processes for obtaining a genetically modified cell with adesired property.

BACKGROUND OF THE INVENTION

The engineering of cells to express new and valuable products is acentral theme in biotechnology. Such metabolic engineering places anumber of competing demands on the host expression platform including i)the need for high and sustained transgene expression, ii) the assemblyof complicated multigene pathways, iii) scalability allowing eitherhigh-throughput trials or larger production runs, and iv) aneasily-ablated host expressome allowing both gene replacement andoptimised substrate pools for newly-engineered metabolic fluxes.Transient leaf assays have emerged as a versatile expression platformfor metabolic engineering by meeting the first three criteria, namelyhigh and extended periods of transgene expression (Voinnet et al.,2003), multigene engineering and high-throughput trait optimisation(Wood et al., 2009; Petrie et al., 2010), and scaling for largerproduction runs as required for personalised medicines (Bakker et al.,2006).

The sustained over-expression of transgenes in leaf assays depends uponviral suppressor proteins (VSP), also known as silencing suppressors, toblock the host cell silencing apparatus. The most widely used VSP is p19that specifically binds 21 nucleotide small RNA with two nucleotide 3′overhangs (Ye et al., 2003) that are generated by the plant cell inresponse to the foreign transgene or hairpin RNAi. Reports have shownthat although p19 enhances gene expression in leaf assays through itssuppressor activity. This VSP also inhibits the effectiveness of RNAi,making it, it is thought, incompatible with simultaneous gene silencingin the same cell (Voinnet et al., 2003; Johansen and Carrington, 2001).

There is a need for methods which allow the concurrent over-expressionof a transgene and the reduced expression of another gene such as anendogenous gene.

SUMMARY OF THE INVENTION

The present inventors have determined that silencing suppressorpolypeptides can be co-expressed with a double stranded RNA (dsRNA) suchas a hairpin RNA or microRNA precursor and at least partial silencing ofa target gene achieved.

Thus, in a first aspect the present invention provides a eukaryoticcell, preferably a plant cell, comprising,

i) a first exogenous polynucleotide encoding a double stranded RNA(dsRNA) molecule which comprises a first nucleotide sequence which iscomplementary to a region of a target RNA encoded by a firstpolynucleotide of interest, and

ii) a second exogenous polynucleotide encoding a silencing suppressorpolypeptide,

wherein each exogenous polynucleotide is operably linked to one or morepromoters that are capable of directing expression of the polynucleotidein the cell, and wherein the cell comprises the silencing suppressorpolypeptide and the dsRNA molecule or a processed RNA product thereofwhich comprises the first nucleotide sequence and is capable of reducingin the cell the level of the target RNA encoded by the firstpolynucleotide of interest and/or the amount of a protein encoded by thetarget RNA when compared to a corresponding cell lacking the firstexogenous polynucleotide.

As the skilled person would appreciate, the silencing suppressorpolypeptide will be compatible with the dsRNA molecule, so that both canexert their effects in the same cell for at least some of the same time.Thus, in the invention the silencing suppressor exerts its suppressiveeffect by a mechanism that allows the double stranded RNA or theprocessed RNA product thereof to reduce the expression of the target RNAand/or reduce the production of a protein encoded by the target RNA.

In an embodiment, the cell further comprises the first polynucleotide ofinterest, wherein the cell has a reduced level of the target RNA encodedby the first polynucleotide of interest and/or a reduced amount of theprotein encoded by the target RNA when compared to a corresponding celllacking the first exogenous polynucleotide. The first polynucleotide ofinterest may not be present in the cell all of the time, for example ifthe first polynucleotide of interest is a gene of a pathogen of the cellsuch as a viral pathogen. In such cases, the dsRNA molecule may beproduced in the cell prior to, or to protect the cell against, thepresence of the first polynucleotide of interest.

In an embodiment, the first polynucleotide of interest is an endogenousgene of the cell or a transgene and/or a gene of a pathogen, such as avirus, of the cell.

In a particularly preferred embodiment, the cell, preferably a plantcell, further comprises a third exogenous polynucleotide, different tothe first and second exogenous polynucleotides and the firstpolynucleotide of interest, which encodes an RNA of interest, such thatin the cell the level of the RNA of interest and/or the amount ofprotein encoded by the RNA of interest is increased when compared to acorresponding cell having the third exogenous polynucleotide and lackingthe second exogenous polynucleotide. That is, it is intended that thethird exogenous polynucleotide is expressed at an increased level in thecell by the presence of the silencing suppressor. The cell may comprisea fourth, fifth or more exogenous polynucleotides which are similarlyexpressed at an increased level by the presence of the silencingsuppressor.

In an embodiment, the first and second exogenous polynucleotides formpart of the same DNA construct, which is preferably integrated into thegenome of the cell.

In a further embodiment, the first, second and third exogenouspolynucleotides form part of the same DNA construct, which is preferablyintegrated into the genome of the cell.

Preferably, at least the second exogenous polynucleotide is integratedinto the genome of the cell.

In an embodiment, the cell, preferably a plant cell, comprises at leasta 25%, preferably at least a 50%, at least a 60%, at least a 70%, atleast an 80%, at least a 90%, at least a 95% reduction in the level ofthe target RNA encoded by the first polynucleotide of interest and/oramount of protein encoded by the target RNA when compared to acorresponding cell lacking the first exogenous polynucleotide. Inanother embodiment, the cell, preferably a plant cell, comprises anabout 25% to about 100%, about 50% to about 100%, about 75% to about100%, about 25% to about 90%, about 50% to about 90%, or about 75% toabout 90% reduction in the level of the target RNA encoded by the firstpolynucleotide of interest and/or amount of protein encoded by thetarget RNA when compared to a corresponding cell lacking the firstexogenous polynucleotide. The extent to which the target RNA is reducedcan be modulated to a desired level by the structure or level of thedsRNA molecule, as desired.

In an embodiment, the silencing suppressor preferentially binds to adouble-stranded RNA molecule which has overhanging 5′ ends relative to acorresponding double-stranded RNA molecule having blunt ends. This is afeature of the V2 type of silencing suppressor, namely for V2 and itsfunctional orthologs. In a further embodiment, the silencing suppressorcomprises amino acids having a sequence as provided in any one of SEQ IDNOs:1, or 38 to 51, a biologically active fragment thereof, or an aminoacid sequence which is at least 30% identical to any one or more of SEQID NOs:1, or 38 to 51.

In another embodiment, the silencing suppressor preferentially binds adsRNA molecule which is 21 base pairs in length relative to a dsRNAmolecule of a different length. This is a feature of at least the p19type of silencing suppressor, namely for p19 and its functionalorthologs. In yet a further embodiment, the silencing suppressorcomprises amino acids having a sequence as provided in any one of SEQ IDNOs: 2, or 27 to 37, a biologically active fragment thereof, or an aminoacid sequence which is at least 30% identical to any one or more of SEQID NOs: 2, or 27 to 37.

In an embodiment, the dsRNA molecule, or a processed RNA productthereof, comprises at least 19 consecutive nucleotides, preferably whoselength is 19-24 nucleotides with 19-24 consecutive basepairs in the caseof a double-stranded hairpin RNA molecule or processed RNA product, morepreferably consisting of 20, 21, 22, 23 or 24 nucleotides in length, andpreferably comprising a methylated nucleotide, which is at least 95%identical to the complement of the region of the target RNA, and whereinthe region of the target RNA is i) within a 5′ untranslated region ofthe target RNA, ii) within a 5′ half of the target RNA, iii) within aprotein-encoding open-reading frame of the target RNA, iv) within a 3′half of the target RNA, or v) within a 3′ untranslated region of thetarget RNA.

In an embodiment, the dsRNA molecule is a microRNA (miRNA) precursorand/or wherein the processed RNA product thereof is a miRNA. Thehybridising sequences in a miRNA precursor are not fully basepaired,having more than one non-basepaired nucleotides in each of thehybridising sequences, which form bulges in the hybridised dsRNAstructure. The basepairing may include one or more G:U basepairs.

In an embodiment, the third exogenous polynucleotide encodes a proteinor microRNA precursor.

In a further embodiment, the cell, preferably a plant cell, furthercomprises at least one, at least two, at least three, at least four orat least five additional, different exogenous polynucleotides eachencoding different RNAs of interest, preferably where the additionalpolynucleotides form part of the same DNA construct.

In an embodiment, the cell, preferably a plant cell, further comprisesat least one, at least two, at least three, at least four or at leastfive additional, different exogenous polynucleotides each independentlyencoding different dsRNA molecules which comprise different nucleotidesequences which are complementary to a region of different target RNAsencoded by different polynucleotides of interest, and/or differentnucleotide sequences which are complementary to different regions of thesame target RNA, preferably where the additional polynucleotides formpart of the same DNA construct.

In an embodiment, the first exogenous polynucleotide encodes more thanone miRNA, preferably at least three, at least four or at least fivemiRNAs, each of which independently comprise different nucleotidesequences which are complementary to a region of different target RNAsencoded by different polynucleotides of interest, and/or differentnucleotide sequences which are complementary to different regions of thesame target RNA. The multiple miRNAs are preferably transcribed from thefirst exogenous polynucleotide as a single miRNA precursor transcriptwhich is subsequently processed into the different miRNAs by thecellular machinery such as a Dicer.

Examples of a eukaryotic cell of the invention include, but are notlimited to, a plant cell, a fungal cell such as a yeast cell, aninvertebrate animal cell, or a vertebrate animal cell. The vertebrateanimal cell may be a mammalian cell such as a human cell or a non-humanmammalian cell. The cell may be in vitro such as in cell culture, or exvivo or in vivo. The cell may be comprised in a tissue, organ ororganism.

In an embodiment, when the cell is a plant cell it is preferably a cellin a plant or in a plant part such as a seed, leaf or stem. The cell maybe of an angiosperm plant, a monocotyledonous plant or a dicotyledonousplant.

In an embodiment, one or more or all of the exogenous polynucleotidesare not integrated into the genome of the cell, i.e. are separate fromthe genome. In this embodiment, the exogenous polynucleotides may beexpressed transiently. The exogenous polynucleotides may lack structuresor sequences required for integration or for replication in the cell.

In an embodiment, the exogenous polynucleotides are operably linked todifferent promoters. In an alternate embodiment, the exogenouspolynucleotides are each operably linked to the same promoter i.e. thesame promoter sequence is used to express each exogenous polynucleotide.In yet a further embodiment, the cell comprises at least three exogenouspolynucleotides where at least two of the promoters are the same and atleast one is different.

In a particularly preferred embodiment, when stably integrated into thegenome the promoter operably linked to the second exogenouspolynucleotide encoding a silencing suppressor polypeptide is not aconstitutive promoter. For example, it is preferred the promoter is atissue specific and/or stage-specific promoter such as a seed-specificpromoter, endosperm-specific promoter, or plant embryo-specificpromoter, or alternatively an inducible promotor. In this embodiment,the promoter is preferentially expressed in the desired tissue or organrelative to other tissues or organs in the organism.

The cells of the invention can be used to modify a wide range ofphenotypes of the cell. For example, wherein expression of the first andsecond exogenous polynucleotides results in modification of fatty acidor carbohydrate synthesis such as starch synthesis in the cell, or ofanother metabolite in the cell. As another example, expression of thefirst, second and third exogenous polynucleotides results inmodification of fatty acid or carbohydrate synthesis such as starchsynthesis in the cell.

In another example, the third exogenous polynucleotide encodes anantibody or an antigen, preferably where expression of the dsRNAmolecule results in a reduction in the level and/or modifies thecomposition of carbohydrates bound to the antibody or antigen. This maybe achieved by the dsRNA molecule by reducing the expression of genes inthe cell which encode glycosyl-, fucosyl- or xylosyl-transferases whichmodify the composition of the carbohydrates.

In another aspect, the present invention provides a plant or a plantpart comprising,

i) a first exogenous polynucleotide encoding a double stranded RNA(dsRNA) molecule which comprises a first nucleotide sequence which iscomplementary to a region of a target RNA encoded by a firstpolynucleotide of interest, and

ii) a second exogenous polynucleotide encoding a silencing suppressorpolypeptide,

wherein each exogenous polynucleotide is operably linked to one or morepromoters that are capable of directing expression of the polynucleotidein a plant cell, and wherein the plant or plant part comprises thesilencing suppressor polypeptide and the dsRNA molecule or a processedRNA product thereof which comprises the first nucleotide sequence and iscapable of reducing in the cell the level of the target RNA encoded bythe first polynucleotide of interest and/or the amount of a proteinencoded by the target RNA when compared to a corresponding cell lackingthe first exogenous polynucleotide. The plant may be angiosperm, amonocotyledonous plant or a dicotyledonous plant, or a part thereof. Theplant part, preferably a seed, may be modified so that it cannotgerminate or give rise to progeny plants. For example, the plant partmay be processed by polishing, milling, grinding or the like.

In an embodiment, the plant or plant part is further characterised byone or more of the above features.

In a further aspect, the present invention provides a process forproducing a eukaryotic cell, preferably a plant cell, of the invention,the method comprising

a) introducing one or more of the exogenous polynucleotides into aeukaryotic cell such that the cell comprises the exogenouspolynucleotides, and

b) expressing the exogenous polynucleotides in the cell.

The cell into which the one or more of the exogenous polynucleotides areintroduced may already have comprised an exogenous polynucleotide otherthan the one or more exogenous polynucleotides, or it may have beennon-transgenic prior to the introduction. This process may be used as ascreening assay to determine whether one or more of the exogenouspolynucleotides have a desired function, or whether the combination ofexogenous polynucleotides together produces a desired phenotype.

In an embodiment, the cell comprises at least the first, second andthird exogenous polynucleotides and the process further comprises one ormore steps selected from:

c) analysing the cell for the presence of one or more of the first,second and third exogenous polynucleotides, the first polynucleotide ofinterest or the RNA of interest, and

d) analysing the cell for a reduction in the level of the target RNAencoded by the first polynucleotide of interest and/or amount of theprotein encoded by the target RNA,

e) analysing the cell for the level of the RNA of interest and/or theamount of protein encoded by the RNA of interest, if present, and

f) selecting a cell which has an increased level of the RNA of interestand/or an increased amount of protein encoded by the RNA of interestwhen compared to a corresponding cell having the third exogenouspolynucleotide and lacking the second exogenous polynucleotide, and/orwhich has a reduced level of the target RNA encoded by the firstpolynucleotide of interest and/or a reduced amount of the proteinencoded by the target RNA when compared to a corresponding cell lackingthe first exogenous polynucleotide.

In an embodiment, step a) comprises introducing into the cell two orthree exogenous polynucleotides.

In an embodiment, the selected cell is further characterised by one ormore of the above features.

In an embodiment, the cell is a plant cell and the process furthercomprises the step of regenerating a transformed plant from a cellcomprising the exogenous polynucleotides. The process may furthercomprise harvesting a plant part, preferably one or more of seed,leaves, stems or tubers, from the transformed plant, and/or obtainingprogeny plants from the transformed plant. The analysing steps asoutlined above may be carried out on the harvested plant part or theprogeny plant.

In an embodiment, the exogenous polynucleotide(s) are expressedtransiently in the cell.

In an embodiment, the cell is a leaf cell in a plant or a cell in aseed.

Also provided is a process for selecting a eukaryotic cell, preferably aplant cell, with a desired property resulting from an increased level ofan RNA of interest and/or amount of protein encoded by the RNA ofinterest, and a reduced level of target RNA encoded by a firstpolynucleotide of interest and/or amount of the protein encoded by thetarget RNA, the process comprising;

i) obtaining one or more cells of the invention comprising the thirdexogenous polynucleotide,

ii) analysing the cell(s) for the desired property,

iii) if the cell(s) does not have the desired property, substituting oneor more of the exogenous polynucleotides with an alternatepolynucleotide(s) and analysing the resultant cell(s) for the desiredproperty,

iv) if necessary, repeating step iii) until the desired property isobtained, and

v) selecting a cell with the desired property.

The cell(s) may be in a tissue, organ or organism, for example in atransgenic plant, such that the analysis of step ii) is carried out atthe level of the tissue, organ or organism. The desired property may beany phenotype of the cell, tissue, organ or organism.

In an embodiment, the first exogenous polynucleotide is substituted suchthat a dsRNA molecule encoded thereby comprises more or less of anucleotide sequence which is closer to the 3′ end of the target RNA whencompared to the exogenous polynucleotide used in the previous step.

In an embodiment, the second exogenous polynucleotide is substitutedwith a different exogenous polynucleotide which encodes a differentsilencing suppressor or candidate silencing suppressor. In thisembodiment, candidate silencing suppressors may be evaluated for theirability to suppress silencing and thereby increase expression of thethird exogenous polynucleotide, or candidate silencing suppressors maybe compared.

In a further aspect, the present invention provides a process forselecting a eukaryotic cell, preferably a plant cell, with a desiredproperty resulting from an increased level of an RNA of interest and/oramount of protein encoded by the RNA of interest, and a reduced level oftarget RNA encoded by a first polynucleotide of interest and/or amountof the protein encoded by the target RNA, the process comprising;

i) obtaining a population of cells, preferably each in a tissue, organor organism such as a transgenic plant, comprising the third exogenouspolynucleotide, and wherein at least some of the cells have differentcombinations of different first, second or third exogenouspolynucleotides,

ii) screening the cell(s) for the desired property, and

iii) selecting one or more cells with the desired property.

In yet another aspect, the present invention provides a process forselecting a eukaryotic cell, preferably a plant cell, with a desiredlevel of silencing of a polynucleotide of interest, the processcomprising;

i) obtaining one or more cells, preferably each in a tissue, organ ororganism such as a transgenic plant, each comprising a first exogenouspolynucleotide encoding a double stranded RNA (dsRNA) molecule whichcomprises a first nucleotide sequence which is complementary to a regionof a target RNA encoded by the polynucleotide of interest, and a secondexogenous polynucleotide encoding a silencing suppressor polypeptide,

ii) analysing the cell(s) for one or more of (a) the level of the targetRNA encoded by the polynucleotide of interest, (b) the amount of theprotein encoded by the target RNA, (c) the level of the dsRNA moleculeor a processed RNA product thereof which comprises the first nucleotidesequence and which is capable of reducing in the cell the level of thetarget RNA encoded by the first polynucleotide of interest and/or theamount of a protein encoded by the target RNA when compared to acorresponding cell lacking the first exogenous polynucleotide, and (d)for a phenotype that is determined by the polynucleotide of interest,

iii) if the cell(s) does not have the desired level of silencing of thepolynucleotide of interest, substituting one or both of the exogenouspolynucleotides with an alternate polynucleotide(s) and analysing theresultant cell(s) for the desired level of silencing,

iv) if necessary, repeating step iii) until the desired level ofsilencing of the polynucleotide of interest is obtained, and

v) selecting a cell with the desired level of silencing,

wherein each exogenous polynucleotide is operably linked to one or morepromoters that are capable of directing expression of thepolynucleotides in the cell. The one or more cells may be analysed atthe same time, in batches, or sequentially. The desired level ofsilencing of the polynucleotide of interest may be assessed by analysingthe cell for a desired property expected as a result of the silencing.

In another aspect, the present invention provides a process forselecting a silencing suppressor which is compatible with adouble-stranded RNA (dsRNA) molecule of interest, comprising the stepsof

i) obtaining one or more eukaryotic cells, preferably plants cells, andpreferably each in a tissue, organ or organism such as a transgenicplant, each of which comprises (a) a first exogenous polynucleotideencoding the dsRNA molecule which comprises a first nucleotide sequencewhich is complementary to a region of a target RNA encoded by a firstpolynucleotide of interest, and (b) a second exogenous polynucleotideencoding a candidate silencing suppressor polypeptide which may becompatible with the dsRNA molecule, wherein each exogenouspolynucleotide is operably linked to one or more promoters that arecapable of directing expression of the polynucleotide in the cell,

ii) analysing the cell(s) for one or more of (a) the level of the targetRNA encoded by the polynucleotide of interest, (b) the amount of theprotein encoded by the target RNA, (c) the level of the dsRNA moleculeor a processed RNA product thereof which comprises the first nucleotidesequence and which is capable of reducing in the cell the level of thetarget RNA encoded by the first polynucleotide of interest and/or theamount of a protein encoded by the target RNA when compared to acorresponding cell lacking the first exogenous polynucleotide, and (d)for a phenotype that is determined by the polynucleotide of interest,

iii) if the cell(s) does not have a desired property, substituting thesecond exogenous polynucleotide in the cell with an alternatepolynucleotide(s) which encodes a candidate compatible silencingsuppressor and repeating step ii), and

iv) if necessary, repeating step iii) until a cell(s) with the desiredproperty is identified, thereby selecting the silencing suppressor whichis compatible with the dsRNA molecule. This process thereby provides ascreening assay to determine whether the silencing suppressor and dsRNAmolecule can both function in the same cell, i.e are compatible, andallows multiple combinations thereof to be tested or compared.

In a preferred embodiment, the cell(s) further comprises a thirdexogenous polynucleotide, different to the first and second exogenouspolynucleotides and the first polynucleotide of interest, and theprocess further comprises the step of analysing the cell(s) for one ormore of the level of expression of the third exogenous polynucleotide ora phenotype of the cell(s) determined by the third exogenouspolynucleotide.

In a more preferred embodiment, the level of expression of the thirdexogenous polynucleotide is increased in the presence of the selectedsilencing suppressor when compared to a corresponding cell having thethird exogenous polynucleotide and lacking the second exogenouspolynucleotide.

In another aspect, the present invention provides a DNA constructcomprising,

i) a first polynucleotide encoding a double stranded RNA (dsRNA)molecule which comprises a first nucleotide sequence which iscomplementary to a region of a target RNA encoded by a firstpolynucleotide of interest, and

ii) a second polynucleotide encoding a silencing suppressor,

wherein each polynucleotide is operably linked to one or more promotersthat are capable of directing expression of the polynucleotides in acell, preferably a plant cell, and the first and second polynucleotidesare exogenous to the cell.

In an embodiment, the DNA construct further comprises a thirdpolynucleotide, different to the first or second polynucleotides orfirst polynucleotide of interest, such that expression of the thirdpolynucleotide in a eukaryotic cell comprising the DNA construct isincreased when compared to a corresponding cell having the thirdpolynucleotide and lacking the second polynucleotide.

In another aspect, the present invention provides a vector comprisingthe DNA construct of the invention.

In a further aspect, the present invention provides a cell comprisingthe DNA construct of the invention and/or the vector of the invention.

Also provided is a cell produced or selected using the process of theinvention.

In another aspect, provided is a transgenic non-human eukaryoticorganism comprising a cell of the invention.

In an embodiment, the transgenic non-human eukaryotic organism is aplant.

In a further aspect, the present invention provides a part of atransgenic non-human eukaryotic organism of the invention comprising acell of the invention.

In an embodiment, the part is a seed, leaf, stem, flower, root or tuber.

The cells, or transgenic non-human organisms comprising the cell or apart thereof, of the invention can be used for a wide variety ofpurposes depending on the cells, the dsRNA and the RNA of interest.Thus, in a further aspect the present invention provides a method ofmaking a product, the method comprising one or more of obtaining,growing, cultivating or culturing a cell of the invention, a transgenicnon-human organism comprising the cell or a part thereof, and optionallyprocessing the cell, organism or part to produce the product.

In an embodiment, the product is one or more of a feedstuff, an oil, afatty acid, a medicament, fuel or an industrial product. The inventionfurther provides for uses of the polynucleotides or cells of theinvention to produce such products.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only.

Functionally-equivalent products, compositions and methods are clearlywithin the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 : V2 allows overexpression of transgenes and their efficientsilencing via hairpin RNAi. A, Time course of GFP expression with eitherno co-infiltrated VSP or the addition of V2 or p19. Image shows onerepresentative leaf photographed up to 7 days post infiltration (dpi),and the image at 7 dpi is used to illustrate the labelling of eachinfiltration zone. B, Time course of the effect of V2 or p19 onhairpin-based silencing of GFP. The image shows one representative leafphotographed at 5 dpi.

FIG. 2 : Western blot analysis of GFP expression in leaves sampled at 4dpi. Image shows one experiment from a duplicate conducted on differentleaves.

FIG. 3 : Analysis of the composition of the phosphatidylcholine (PC)fraction of leaves infiltrated with various combinations of V2, p19 andhpNbFAD2. Leaves were sampled 5 dpi and the error bars represent thestandard error of the mean from 5 independent leaves.

FIG. 4 : Analysis of the content and composition of leaf oils whenleaves were infiltrated with combinations of V2, p19, hpNbFAD2, DGAT1and oleosin. Leaves were samples 5 dpi and error bars represent thestandard error of the mean calculated from 5 independent leaves.

FIG. 5 : The enzymatic production of DHS from oleic acid.

FIG. 6 : Comparison of the production of DHS in leaf assays using eitherEcCPFAS or GhCPFAS in transient leaf assays. Leaves were harvested 4dpi.

FIG. 7 : Overexpression of transgenes and silencing of an endogene forimproved fluxes of DHS into leaf oils. Leaves were harvested 4 dpi.These comparisons were conducted on 4 different leaves, and this figureshows results from one representative leaf.

FIG. 8 : ‘Deep sequencing’ analysis of the size and distribution ofsmall RNA populations generated by a hairpin targeting the endogeneNbFAD2. The full-length NbFAD2 is portrayed indicating the region usedto generate a 660 bp hairpin, hpNbFAD2. The size and distribution of thedominate classes of small RNAs on the forward (F) and reverse (R) strandof the NbFAD2 is illustrated below. Each track is rescaled to show therelatively uneven distribution of small RNAs across the target.

FIG. 9 : Absolute numbers of the dominant small RNA size classesgenerated by hpNbFAD2. The relative percentage of each size class isgiven in the text.

FIG. 10 : DHS is accumulated in leaf oils.

FIG. 11 : Fatty acid profile of leaves producing DHS in the presence orabsence of the elongase AtFAE1. Elongation experiments were conducted on3 different leaves, and the figure shows a representative fatty acidprofile from a single leaf.

FIG. 12 : The identification of eDHS using a range of GC and MStechniques. The upper panels show GC (FID) traces for lipid extractsfrom leaves infiltrated with the combination of genes as shown. Commonand new metabolites are shown as indicated. Lower panels show the rangeof masses for metabolites first resolved on the GC, DHS and eDHS. Theinserts for each MS indicates the structure of DHS and eDHS.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1—amino acid sequence of tomato leaf yellow curl virus V2protein

SEQ ID NO: 2—amino acid sequence of tomato bushy stunt virus P19 protein

SEQ ID NO: 3—nucleotide sequence encoding tomato leaf yellow curl virusV2 protein

SEQ ID NO: 4—nucleotide sequence encoding tomato bushy stunt virus P19protein

SEQ ID NO: 5—amino acid sequence of Sesmum indicum oleosin protein

SEQ ID NO: 6—nucleotide sequence encoding Sesmum indicum oleosin protein

SEQ ID NO: 7—amino acid sequence of Arabidopsis thaliana AtFAE1 protein

SEQ ID NO: 8—nucleotide acid sequence encoding Arabidopsis thalianaAtFAE1 protein including 5′ intron sequence

SEQ ID NO: 9—amino acid sequence of Arabidopsis thaliana AtDGAT1 protein

SEQ ID NO: 10—nucleotide acid sequence encoding Arabidopsis thalianaAtDGAT1 protein

SEQ ID NO: 11—amino acid sequence of NbFAD2 protein

SEQ ID NO: 12—nucleotide sequence encoding dsRNA hairpin targeting N.benthamiana FAD2

SEQ ID NOs 13 to 20—oligonucleotide primers

SEQ ID NO: 21—amino acid sequence of Gossypium hirsutum CPFAS-1(truncated protein)

SEQ ID NO: 22—amino acid sequence of Gossypium hirsutum CPFAS-1

SEQ ID NO: 23—nucleotide sequence encoding truncated Gossypium hirsutumCPFAS-1

SEQ ID NO: 23—nucleotide sequence encoding Gossypium hirsutum CPFAS-1

SEQ ID NO: 24—amino acid sequence of Escherichia coli CPFAS

SEQ ID NO: 26—codon optimized E. coli CPFAS open reading frame for plantexpression

SEQ ID NO: 27—Cymbidium ringspot tombus virus p19 like silencingsuppressor

SEQ ID NO: 28—Pelargonium necrotic spot virus p19 like silencingsuppressor

SEQ ID NO: 29—Havel river tombus virus p19 like silencing suppressor

SEQ ID NO: 30—Cucumber necrosis virus p19 like silencing suppressor

SEQ ID NO: 31—Grapevine Algerian latent virus p19 like silencingsuppressor

SEQ ID NO: 32—Pear latent virus p19 like silencing suppressor

SEQ ID NO: 33—Lisianthus necrotic virus p19 like silencing suppressor

SEQ ID NO: 34—Lettuce necrotic stunt virus p19 like silencing suppressor

SEQ ID NO: 35—Artichoke Mottled Crinkle virus p19 like silencingsuppressor

SEQ ID NO: 36—Carnation Italian ringspot virus p19 like silencingsuppressor

SEQ ID NO: 37—Maize necrotic steak virus virus p19 like silencingsuppressor

SEQ ID NO: 38—Watermelon chlorotic stunt virus V2 like silencingsuppressor

SEQ ID NO: 39—Okra yellow wrinkle virus V2 like silencing suppressor

SEQ ID NO: 40—Okra leaf curl virus V2 like silencing suppressor

SEQ ID NO: 41—Tomato leaf curl togo virus V2 like silencing suppressor

SEQ ID NO: 42—Ageratum leaf curl Cameroon virus V2 like silencingsuppressor

SEQ ID NO: 43—East African cassava mosaic Malawi virus V2 like silencingsuppressor

SEQ ID NO: 44—South African cassava mosaic virus V2 like silencingsuppressor

SEQ ID NO: 45—Tomato leaf curl Madagascar virus V2 like silencingsuppressor

SEQ ID NO: 46—Tomato leaf curl Zimbabwe virus V2 like silencingsuppressor

SEQ ID NO: 47—Tomato begomovirus V2 like silencing suppressor

SEQ ID NO: 48—Tomato leaf curl Namakely virus V2 like silencingsuppressor

SEQ ID NO: 49—Pepper yellow vein Mali virus V2 like silencing suppressor

SEQ ID NO: 50—Tomato leaf curl Sudan virus V2 like silencing suppressor

SEQ ID NO: 51—Tomato leaf curl Oman virus V2 like silencing suppressor

SEQ ID NO: 52—nucleotide sequence encoding miRNA targeting A. thalianapytoene desaturase

SEQ ID NO: 53—nucleotide sequence encoding miRNA targeting A. thalianaFAD2

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term “about”, unless stated to the contrary, refersto +/−20%, more preferably +/−10%, more preferably +/−5%, morepreferably +/−2%, more preferably +/−1%, of the designated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The term “exogenous” in the context of a polynucleotide or polypeptiderefers to the polynucleotide or polypeptide when present in a cell thatdoes not naturally comprise the polynucleotide or polypeptide. In anembodiment, the exogenous polynucleotide or polypeptide is from adifferent genus. In another embodiment, the exogenous polynucleotide orpolypeptide is from a different species. In one embodiment the exogenouspolynucleotide or polypeptide is expressed in a host organism or celland the exogenous polynucleotide or polypeptide is from a differentspecies or genus.

The term “corresponding” refers to a cell, or non-human eukaryoticorganism or part thereof that has the same or similar genetic backgroundas a cell, or non-human eukaryotic organism or part thereof of theinvention but that has not been modified as described herein. Forexample, the cell, or non-human eukaryotic organism or part thereoflacks the first exogenous polynucleotide encoding the dsRNA, and/orwhich lacks the second exogenous polynucleotide encoding the silencingsuppressor polypeptide. A corresponding cell or non-human eukaryoticorganism or part thereof can be used as a control to comparelevels/amount of, for example, RNA and/or protein, or the extent andnature of trait modification, for example non-polar lipid or starchproduction and/or content, with a cell, or non-human eukaryotic organismor part thereof modified as described herein. A person skilled in theart is able to readily determine an appropriate “corresponding” cell,tissue, organ or organism for such a comparison.

As used herein “compared to”, “relative to” or variations thereof refersto comparing, for example, the levels/amount of RNA and/or protein ofthe transgenic cell, or non-human eukaryotic organism or part thereof,expressing the one or more exogenous polynucleotides with a cell, ornon-human eukaryotic organism or part thereof lacking the one or moreexogenous polynucleotides.

The term “transgenic non-human eukaryotic organism” refers to, forexample, a whole plant, algae, non-human animal, or an organism suitablefor fermentation such as a yeast or fungus, comprising an exogenouspolynucleotide (transgene). In one embodiment, the transgenic non-humanorganism is a phototrophic organism (for example, a plant or alga)capable of obtaining energy from sunlight to synthesize organiccompounds for nutrition.

As used herein, a “desired property” refers to a phenotype which is notpossessed by the cell but which is desired. The property may be anincrease or decrease (or abolished) in the level of an existingphenotype or a phenotype not possessed by the cell without the exogenouspolynucleotides.

Silencing Suppressors

Post-transcriptional gene silencing (PTGS) is a nucleotidesequence-specific defense mechanism that can target both cellular andviral mRNAs for degradation. PTGS occurs in eukaryotic cells such asplants or fungi stably or transiently transformed with a recombinantpolynucleotide(s) and results in the reduced accumulation of RNAmolecules with sequence similarity to the introduced polynucleotide.“Post-transcriptional” refers to a mechanism for the reduction operatingat least partly, but not necessarily exclusively, after production of aninitial RNA transcript, for example during processing of the initial RNAtranscript, or concomitant with splicing or export of the RNA to thecytoplasm, or within the cytoplasm by complexes associated withArgonaute proteins.

RNA molecule levels can be increased, and/or RNA molecule levelsstabilized over numerous generations or under different environmentalconditions, by limiting the expression of a silencing suppressor in astorage organ of a plant or part thereof. As used herein, a “silencingsuppressor” is any polypeptide that can be expressed in a eukaryoticcell that enhances the level of expression product from a differenttransgene in the cell, particularly, over repeated generations from theinitially transformed cell.

In an embodiment, the silencing suppressor is a viral silencingsuppressor or mutant thereof. A large number of viral silencingsuppressors are known in the art and include, but are not limited toP19, V2 (Glick et al. 2008; Fukunaga and Doudna, 2009), P38, Pe-Po andRPV-P0. Examples of suitable viral silencing suppressors include thosedescribed in WO 2010/057246.

A silencing suppressor may be stably expressed in a transgenic non-humaneukaryotic organism or part thereof of the present invention. As usedherein, the term “stably expressed” or variations thereof refers to thelevel of the RNA molecule being essentially the same or higher inprogeny cells, organisms or parts over repeated generations, forexample, at least three, at least five, or at least ten generations,when compared to corresponding cells, organisms or parts lacking theexogenous polynucleotide encoding the silencing suppressor. However,this term(s) does not exclude the possibility that over repeatedgenerations there is some loss of levels of the RNA molecule whencompared to a previous generation, for example, not less than a 10% lossper generation.

The suppressor can be selected from any source e.g. plant, viral,mammal, etc. The suppressor may be, for example:

flock house virus B2,

pothos latent virus P14,

pothos latent virus AC2,

African cassava mosaic virus AC4,

bhendi yellow vein mosaic disease C2,

bhendi yellow vein mosaic disease C4,

bhendi yellow vein mosaic disease βC1,

tomato chlorosis virus p22,

tomato chlorosis virus CP,

tomato chlorosis virus CPm,

tomato golden mosaic virus AL2,

tomato leaf curl Java virus βC1,

tomato yellow leaf curl virus V2,

tomato yellow leaf curl virus-China C2,

tomato yellow leaf curl China virus Y10 isolate βC1,

tomato yellow leaf curl Israeli isolate V2,

mungbean yellow mosaic virus-Vigna AC2,

hibiscus chlorotic ringspot virus CP,

turnip crinkle virus P38,

turnip crinkle virus CP,

cauliflower mosaic virus P6,

beet yellows virus p21,

citrus tristeza virus p20,

citrus tristeza virus p23,

citrus tristeza virus CP,

cowpea mosaic virus SCP,

sweet potato chlorotic stunt virus p22,

cucumber mosaic virus 2b,

tomato aspermy virus HC-Pro,

beet curly top virus L2,

soil borne wheat mosaic virus 19K,

barley stripe mosaic virus Gammab,

poa semilatent virus Gammab,

peanut clump pecluvirus P15,

rice dwarf virus Pns10,

curubit aphid borne yellows virus P0,

beet western yellows virus P0,

potato virus X P25,

cucumber vein yellowing virus P1b,

plum pox virus HC-Pro,

sugarcane mosaic virus HC-Pro,

potato virus Y strain HC-Pro,

tobacco etch virus P1/HC-Pro,

turnip mosaic virus P1/HC-Pro,

cocksfoot mottle virus P1,

cocksfoot mottle virus-Norwegian isolate P1,

rice yellow mottle virus P1,

rice yellow mottle virus-Nigerian isolate P1,

rice hoja blanca virus NS3,

rice stripe virus NS3,

crucifer infecting tobacco mosaic virus 126K,

crucifer infecting tobacco mosaic virus p122,

tobacco mosaic virus p122,

tobacco mosaic virus 126,

tobacco mosaic virus 130K,

tobacco rattle virus 16K,

tomato bushy stunt virus P19,

tomato spotted wilt virus NSs,

apple chlorotic leaf spot virus P50,

grapevine virus A p10,

grapevine leafroll associated virus-2 homolog of BYV p21,

as well as variants/mutants thereof. The list above provides the virusfrom which the suppressor can be obtained and the protein (e.g., B2,P14, etc.), or coding region designation for the suppressor from eachparticular virus. Other candidate silencing suppressors may be obtainedby examining viral genome sequences for polypeptides encoded at the sameposition within the viral genome, relative to the structure of a relatedviral genome comprising a known silencing suppressor, as is appreciatedby a person of skill in the art.

Silencing suppressors can be categorized based on their mode of action.Suppressors such as V2 which preferentially bind to a double-strandedRNA molecule which has overhanging 5′ ends relative to a correspondingdouble-stranded RNA molecule having blunt ends have been found to beparticularly useful for enhancing transgene expression when used incombination with gene silencing, in particular with the use of anexogenous polynucleotide encoding a dsRNA. Other suppressors such as p19which preferentially bind a dsRNA molecule which is 21 base pairs inlength relative to a dsRNA molecule of a different length can also allowtransgene expression in the presence of an exogenous polynucleotideencoding a dsRNA, but generally to a lesser degree than, for example,V2. This allows the selection of an optimal combination of dsRNA,silencing suppressor and over-expressed transgene for a particularpurpose. Such optimal combinations can be identified using a method ofthe invention.

In an embodiment, the silencing suppressor preferentially binds to adouble-stranded RNA molecule which has overhanging 5′ ends relative to acorresponding double-stranded RNA molecule having blunt ends. In thiscontext, the corresponding double-stranded RNA molecule preferably hasthe same nucleotide sequence as the molecule with the 5′ overhangingends, but without the overhanging 5′ ends. Binding assays are routinelyperformed, for example in in vitro assays, by any method as known to aperson of skill in the art.

In a further embodiment, the silencing suppressor comprises amino acidshaving a sequence as provided in any one of SEQ ID NOs:1, or 38 to 51, abiologically active fragment thereof, or an amino acid sequence which isat least 30% identical to any one or more of SEQ ID NOs:1, or 38 to 51.

Multiple copies of a suppressor may be used. Different suppressors maybe used together (e.g., in tandem).

Essentially any RNA molecule of interest which is desirable to beexpressed in a cell, organism or part can be co-expressed with thesilencing suppressor. The RNA molecule may influence, for example, anagronomic trait, insect resistance, disease resistance, herbicideresistance, sterility, grain characteristics, and the like. The encodedpolypeptides may be involved in metabolism of lipid, starch,carbohydrates, nutrients, etc., or may be responsible for the synthesisof proteins, peptides, lipids, waxes, starches, sugars, carbohydrates,flavors, odors, toxins, carotenoids, hormones, polymers, flavonoids,storage proteins, phenolic acids, alkaloids, lignins, tannins,celluloses, glycoproteins, glycolipids, etc.

In a particular example, the plants produce increased levels of enzymesfor lipid production in plants such as Brassicas, for example oilseedrape or sunflower, safflower, flax, cotton, soybean or maize.

Silencing

As used herein, the term “a double stranded RNA (dsRNA) molecule whichcomprises a first nucleotide sequence which is complementary to a regionof a target RNA encoded by a first polynucleotide of interest” orvariations thereof refers to an RNA molecule which can be used todownregulate the levels of a target RNA, and/or the amount of proteinencoded by the target RNA, in a cell, comprising a double-stranded RNAregion comprising the first nucleotide sequence (“antisense sequence”)and its complement (“sense sequence”). The target RNA, which is encodedby the first polynucleotide of interest which may be an RNA molecule(e.g. a viral RNA molecule) or preferably a DNA molecule which istranscribed (or replicated) in the cell to produce the target RNA, maybe produced by the genome of the cell, or may be produced by a pathogenof the cell such as a virus. Thus, due to temporal and/or spatialexpression patterns of an endogenous gene, or the absence of thepathogen, the dsRNA may not always be present in the cell at the sametime as the target RNA.

As the skilled person would appreciate, to exert the desired effect adsRNA targeting the transcription product of an endogenous gene will beexpressed at least some of the same time as the endogenous gene. Whilst,as described below, the dsRNA may comprise single stranded regions, thedouble stranded region comprises a sequence (antisense sequence) whichis complementary to a region of the target. Typically, the complementaryregion is at least 19 consecutive nucleotides in length, preferably19-30 nucleotides for use in vertebrate animal cells such as mammaliancells, more preferably 19-25 nucleotides, most preferably of 20, 21, 22,23 or 24 nucleotides in length. The complementarity may be partial orcomplete to the region of the target RNA. Partial complementarity,particularly in the context of a target RNA in an animal cell such as avertebrate animal cell or mammalian animal cell preferably includes aregion of at least 6 consecutive nucleotides, preferably at least 7, atleast 8, at least 9, or at least 10 consecutive nucleotides, andpreferably includes consecutive nucleotides 2-8 of the nucleotidesequence counting from the 5′ end.

For plant cells, the complementarity is preferably full complementarityover a region of 19, 20 or 21 consecutive nucleotides, or over a regionof at least 30 nucleotides, at least 50 nucleotides, or at least 100nucleotides when the dsRNA molecule is a hairpin RNA. Complementarity inthe context of this paragraph includes G:U basepairs as well as G:C andA:U basepairs.

RNA Interference

RNA interference (RNAi) is particularly useful for specificallyinhibiting the production of a particular protein or functional RNA.This technology relies on the presence of dsRNA molecules that contain asequence that is essentially identical to the mRNA of the gene ofinterest or part thereof. Conveniently, the dsRNA can be produced from asingle promoter in a recombinant vector or host cell, where the senseand anti-sense sequences are flanked by an unrelated sequence whichenables the sense and anti-sense sequences to hybridize to form thedsRNA molecule with an unrelated sequence forming a loop structure,although a sequence with identity to the target RNA or its complementcan form the loop structure. Typically, the dsRNA is encoded by adouble-stranded DNA construct which has sense and antisense sequences inan inverted repeat structure, arranged as an interrupted palindrome,where the repeated sequences are transcribed to produce the hybridisingsequences in the dsRNA molecule, and the interrupt sequence istranscribed to form the loop in the dsRNA molecule. The design andproduction of suitable dsRNA molecules is well within the capacity of aperson skilled in the art, particularly considering Waterhouse et al.(1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, andWO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology, preferably atleast 19 consecutive nucleotides complementary to a region of, a targetRNA, to be inactivated. The DNA therefore comprises both sense andantisense sequences that, when transcribed into RNA, can hybridize toform the double stranded RNA region. In one embodiment of the invention,the sense and antisense sequences are separated by a spacer region thatcomprises an intron which, when transcribed into RNA, is spliced out.This arrangement has been shown to result in a higher efficiency of genesilencing. The double stranded region may comprise one or two RNAmolecules, transcribed from either one DNA region or two. The presenceof the double stranded molecule is thought to trigger a response from anendogenous system that destroys both the double stranded RNA and alsothe homologous RNA transcript from the target gene, efficiently reducingor eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides. The full-length sequencecorresponding to the entire gene transcript may be used. The degree ofidentity of the sense and antisense sequences to the targeted transcriptshould be at least 85%, at least 90%, or at least 95-100%. The RNAmolecule may of course comprise unrelated sequences which may functionto stabilize the molecule. The RNA molecule may be expressed under thecontrol of a RNA polymerase II or RNA polymerase III promoter. Examplesof the latter include tRNA or snRNA promoters.

Furthermore, it has been established by the inventors that the positionof the complementary sequence relevant to the target, namely withrespect to the 5′ or 3′ end, can influence the level of silencing in thepresence of a silencing suppressor polypeptide. Thus, using a method ofthe invention an optimal combination of dsRNA sequence and silencingsuppressor can be determined on an as needs basis.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the siRNA sequence commenceswith the dinucleotide AA, comprises a GC-content of about 30-70%(preferably, 30-60%, more preferably 40-60% and more preferably about45%-55%), and does not have a high percentage identity to any nucleotidesequence other than the target in the genome of the organism in which itis to be introduced, for example, as determined by standard BLASTsearch.

microRNA

MicroRNAs (abbreviated miRNAs) are generally 19-25 nucleotides (commonlyabout 20-24 nucleotides in plants) non-coding RNA molecules that arederived from larger precursors that form imperfect stem-loop structures.

miRNAs bind to complementary sequences on target messenger RNAtranscripts (mRNAs), usually resulting in translational repression ortarget degradation and gene silencing.

In a preferred embodiment, the miRNA is an artificial (man made) miRNA.In other words, the miRNA is a non-naturally occurring miRNA.

In a further particularly preferred embodiment, if the dsRNA is a miRNAsuch as a miRNA comprising a dsRNA region of 21 base pairs expressed asa precursor miRNA, and the cell further comprises the third exogenouspolynucleotide, the silencing suppressor is a V2-like polypeptide suchas those comprising amino acids having a sequence as provided in any oneof SEQ ID NOs:1, or 38 to 51, a biologically active fragment thereof, oran amino acid sequence which is at least 30% identical to any one ormore of SEQ ID NOs:1, or 38 to 51.

In plant cells, miRNA precursor molecules are believed to be largelyprocessed in the nucleus. The pri-miRNA (containing one or more localdouble-stranded or “hairpin” regions as well as the usual 5′ “cap” andpolyadenylated tail of an mRNA) is processed to a shorter miRNAprecursor molecule that also includes a stem-loop or fold-back structureand is termed the “pre-miRNA”. In plants, the pre-miRNAs are cleaved bydistinct DICER-like (DCL) enzymes, in particular DCL-1, yieldingmiRNA:miRNA* duplexes. Prior to transport out of the nucleus, theseduplexes are methylated. In contrast, hairpin RNA molecules havinglonger dsRNA regions are processed in particular by DCL-3 and DCL-4.Most mammalian cells have only a single DICER polypeptide which cleavesmultiple dsRNA structures.

In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex isselectively incorporated into an active RNA-induced silencing complex(RISC) for target recognition. The RISC-complexes contain a particularsubset of Argonaute proteins that exert sequence-specific generepression (see, for example, Millar and Waterhouse, 2005; Pasquinelliet al., 2005; Almeida and Allshire, 2005).

Cosuppression

Genes can suppress the expression of related endogenous genes and/ortransgenes already present in the genome, a phenomenon termedhomology-dependent gene silencing. Most of the instances of homologydependent gene silencing fall into two classes—those that function atthe level of transcription of the transgene, and those that operatepost-transcriptionally.

Post-transcriptional homology-dependent gene silencing (i.e.,cosuppression) describes the loss of expression of a transgene andrelated endogenous or viral genes in transgenic plants. Cosuppressionoften, but not always, occurs when transgene transcripts are abundant,and it is generally thought to be triggered at the level of mRNAprocessing, localization, and/or degradation. Several models exist toexplain how cosuppression works (see in Taylor, 1997).

One model, the “quantitative” or “RNA threshold” model, proposes thatcells can cope with the accumulation of large amounts of transgenetranscripts, but only up to a point. Once that critical threshold hasbeen crossed, the sequence-dependent degradation of both transgene andrelated endogenous gene transcripts is initiated. It has been proposedthat this mode of cosuppression may be triggered following the synthesisof copy RNA (cRNA) molecules by reverse transcription of the excesstransgene mRNA, presumably by endogenous RNA-dependent RNA polymerases.These cRNAs may hybridize with transgene and endogenous mRNAs, theunusual hybrids targeting homologous transcripts for degradation.However, this model does not account for reports suggesting thatcosuppression can apparently occur in the absence of transgenetranscription and/or without the detectable accumulation of transgenetranscripts.

To account for these data, a second model, the “qualitative” or“aberrant RNA” model, proposes that interactions between transgene RNAand DNA and/or between endogenous and introduced DNAs lead to themethylation of transcribed regions of the genes. The methylated genesare proposed to produce RNAs that are in some way aberrant, theiranomalous features triggering the specific degradation of all relatedtranscripts. Such aberrant RNAs may be produced by complex transgeneloci, particularly those that contain inverted repeats.

A third model proposes that intermolecular base pairing betweentranscripts, rather than cRNA-mRNA hybrids generated through the actionof an RNA-dependent RNA polymerase, may trigger cosuppression. Such basepairing may become more common as transcript levels rise, the putativedouble-stranded regions triggering the targeted degradation ofhomologous transcripts. A similar model proposes intramolecular basepairing instead of intermolecular base pairing between transcripts.Cosuppression involves introducing an extra copy of a gene or a fragmentthereof into a plant in the sense orientation with respect to a promoterfor its expression. A skilled person would appreciate that the size ofthe sense fragment, its correspondence to target gene regions, and itsdegree of sequence identity to the target gene can vary. In someinstances, the additional copy of the gene sequence interferes with theexpression of the target plant gene. Reference is made to WO 97/20936and EP 0465572 for methods of implementing co-suppression approaches.

The present inventors postulate that the V2 silencing suppressor and itsfunctional analogs suppress the co-suppression pathway but not, or to alesser extent, the microRNA and RNA interference pathways as describedabove.

Polynucleotides

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof. Apolynucleotide useful for the invention may be of genomic, cDNA,semisynthetic, or synthetic origin, double-stranded or single-strandedand by virtue of its origin or manipulation: (1) is not associated withall or a portion of a polynucleotide with which it is associated innature, (2) is linked to a polynucleotide other than that to which it islinked in nature, or (3) does not occur in nature. The following arenon-limiting examples of polynucleotides: coding regions which may ormay not include introns, messenger RNA (mRNA), transfer RNA (tRNA),ribosomal RNA (rRNA), cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence, andchimeric DNA including DNA constructs.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals, in which case, thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns”, “interveningregions”, or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (nRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the mRNA transcript. The mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.The term “gene” includes a synthetic or fusion molecule encoding all orpart of the proteins of the invention described herein and acomplementary nucleotide sequence to any one of the above.

As used herein, “chimeric DNA” refers to any DNA molecule that is notnaturally found in nature; also referred to herein as a “DNA construct”.Typically, chimeric DNA comprises regulatory and transcribed or proteincoding sequences that are not naturally found together in nature.Accordingly, chimeric DNA may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. The openreading frame may or may not be linked to its natural upstream anddownstream regulatory elements. The open reading frame may beincorporated into, for example, a plant genome, in a non-naturallocation, or in a replicon or vector where it is not naturally foundsuch as a bacterial plasmid or a viral vector. The term “chimeric DNA”is not limited to DNA molecules which are replicable in a host, butincludes DNA capable of being ligated into a replicon by, for example,specific adaptor sequences.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure. The terms “genetically modified”,“transgenic”, “recombinant” and variations thereof include introducing agene into a cell by transformation or transduction, mutating a gene in acell and genetically altering or modulating the regulation of a gene ina cell, or the progeny of any cell modified as described above.

A “genomic region” as used herein refers to a position within the genomewhere a transgene, or group of transgenes (also referred to herein as acluster), have been inserted into a cell, or predecessor thereof. Suchregions only comprise nucleotides that have been incorporated by theintervention of man such as by methods described herein.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 40%, more preferably at least50%, more preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99%, morepreferably at least 99.1%, more preferably at least 99.2%, morepreferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

A polynucleotide of, or useful for, the present invention mayselectively hybridise, under stringent conditions, to a polynucleotidedefined herein. As used herein, stringent conditions are those that: (1)employ during hybridisation a denaturing agent such as formamide, forexample, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfateat 42° C. in 0.2×SSC and 0.1% SDS, and/or (3) employ low ionic strengthand high temperature for washing, for example, 0.015 M NaCl/0.0015 Msodium citrate/0.1% SDS at 50° C. Shorter polynucleotides, for examplethose of 19-24 nucleotides, require less stringent conditions forhybridisation, as is well understood by persons of skill in the art. Forexample, the hybridisation conditions may omit the formamide, and thewashing conditions use a temperature of 37° C. with higher salt andlower SDS concentrations.

Polynucleotides of the invention may possess, when compared to naturallyoccurring molecules, one or more mutations which are deletions,insertions, or substitutions of nucleotide residues. Polynucleotideswhich have mutations relative to a reference sequence can be eithernaturally occurring (that is to say, isolated from a natural source) orsynthetic (for example, by performing site-directed mutagenesis or DNAshuffling on the nucleic acid).

Expression Vector

As used herein, an “expression vector” is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of oneor more specified polynucleotides. Preferably, the expression vector isalso capable of replicating within the host cell. Expression vectors aretypically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in host cells of the present invention, including in fungal,endoparasite, arthropod, animal, algal, and plant cells. Particularlypreferred expression vectors of the present invention can direct geneexpression in yeast, animal, and/or plant cells.

“Operably linked” as used herein, refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence of a polynucleotide defined herein,if it stimulates or modulates the transcription of the coding sequencein an appropriate cell. Generally, promoter transcriptional regulatoryelements that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

Expression vectors of the present invention contain regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the host cell and that control the expression ofpolynucleotides of the present invention. In particular, expressionvectors of the present invention include transcription controlsequences. Transcription control sequences are sequences which controlthe initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in at leastone of the recombinant cells of the present invention. The choice of theregulatory sequences used depends on the target organism such as a plantand/or target organ or tissue of interest. Such regulatory sequences maybe obtained from any eukaryotic organism such as plants or plantviruses, or may be chemically synthesized. A variety of suchtranscription control sequences are known to those skilled in the art.Particularly preferred transcription control sequences are promotersactive in directing transcription in plants, either constitutively orstage and/or tissue specific, depending on the use of the plant orpart(s) thereof.

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in forexample, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985,supp. 1987, Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 1989, and Gelvin et al., Plant MolecularBiology Manual, Kluwer Academic Publishers, 1990. Typically, plantexpression vectors include for example, one or more cloned plant genesunder the transcriptional control of 5′ and 3′ regulatory sequences anda dominant selectable marker. Such plant expression vectors also cancontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants, see for example, WO 84/02913. All of these promoters havebeen used to create various types of plant-expressible recombinant DNAvectors.

For the purpose of expression in source tissues of the plant such as theleaf, seed, root or stem, it is preferred that the promoters utilized inthe present invention have relatively high expression in these specifictissues. For this purpose, one may choose from a number of promoters forgenes with tissue- or cell-specific, or -enhanced expression. Examplesof such promoters reported in the literature include, the chloroplastglutamine synthetase GS2 promoter from pea, the chloroplastfructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.Also reported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase promoter from eastern larch (Larixlaricina), the promoter for the Cab gene, Cab6, from pine, the promoterfor the Cab-1 gene from wheat, the promoter for the Cab-1 gene fromspinach, the promoter for the Cab 1R gene from rice, the pyruvate,orthophosphate dikinase (PPDK) promoter from Zea mays, the promoter forthe tobacco Lhcb1*2 gene, the Arabidopsis thaliana Suc2 sucrose-H³⁰symporter promoter, and the promoter for the thylakoid membrane proteingenes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD, Cab, RbcS).Other promoters for the chlorophyll a/f-binding proteins may also beutilized in the present invention such as the promoters for LhcB geneand PsbP gene from white mustard (Sinapis alba).

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of RNA-binding protein genes in plant cells,including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3Apromoter, maize RbcS promoter), (3) hormones such as abscisic acid, (4)wounding (e.g., WunI), or (5) chemicals such as methyl jasmonate,salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or itmay also be advantageous to employ (6) organ-specific promoters.

For the purpose of expression in sink tissues of the plant such as thetuber of the potato plant, the fruit of tomato, or the seed of soybean,canola, cotton, Zea mays, wheat, rice, and barley, it is preferred thatthe promoters utilized in the present invention have relatively highexpression in these specific tissues. A number of promoters for geneswith tuber-specific or -enhanced expression are known, including theclass I patatin promoter, the promoter for the potato tuber ADPGPPgenes, both the large and small subunits, the sucrose synthase promoter,the promoter for the major tuber proteins, including the 22 kD proteincomplexes and proteinase inhibitors, the promoter for the granule boundstarch synthase gene (GBSS), and other class I and II patatinspromoters. Other promoters can also be used to express a protein inspecific tissues such as seeds or fruits. The promoter for 0-conglycininor other seed-specific promoters such as the napin, zein, linin andphaseolin promoters, can be used. Root specific promoters may also beused. An example of such a promoter is the promoter for the acidchitinase gene. Expression in root tissue could also be accomplished byutilizing the root specific subdomains of the CaMV 35S promoter thathave been identified.

In one embodiment, the promoter directs expression in tissues and organsin which lipid biosynthesis take place. Such promoters act in seeddevelopment at a suitable time for modifying lipid composition in seeds.

In a further particularly preferred embodiment, the promoter is a plantstorage organ specific promoter. As used herein, the term “plant storageorgan specific promoter” refers to a promoter that preferentially, whencompared to other plant tissues, directs gene transcription in a storageorgan of a plant. Preferably, the promoter only directs expression of agene of interest in the storage organ, and/or expression of the gene ofinterest in other parts of the plant such as leaves is not detectable byNorthern blot analysis and/or RT-PCR. Typically, the promoter drivesexpression of genes during growth and development of the storage organ,in particular during the phase of synthesis and accumulation of storagecompounds in the storage organ. Such promoters may drive gene expressionin the entire plant storage organ or only part thereof such as theseedcoat, embryo or cotyledon(s) in seeds of dicotyledonous plants orthe endosperm or aleurone layer of seeds of monocotyledonous plants.

In one embodiment, the plant storage organ specific promoter is a seedspecific promoter. In a more preferred embodiment, the promoterpreferentially directs expression in the cotyledons of a dicotyledonousplant or in the endosperm of a monocotyledonous plant, relative toexpression in the embryo of the seed or relative to other organs in theplant such as leaves. Preferred promoters for seed-specific expressioninclude: 1) promoters from genes encoding enzymes involved in lipidbiosynthesis and accumulation in seeds such as desaturases andelongases, 2) promoters from genes encoding seed storage proteins, and3) promoters from genes encoding enzymes involved in carbohydratebiosynthesis and accumulation in seeds. Seed specific promoters whichare suitable are, the oilseed rape napin gene promoter (U.S. Pat. No.5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), theArabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgarisphaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter(WO 91/13980), or the legumin B4 promoter (Baumlein et al., 1992), andpromoters which lead to the seed-specific expression in monocots such asmaize, barley, wheat, rye, rice and the like. Notable promoters whichare suitable are the barley lpt2 or lpt1 gene promoter (WO 95/15389 andWO 95/23230), or the promoters described in WO 99/16890 (promoters fromthe barley hordein gene, the rice glutelin gene, the rice oryzin gene,the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene,the maize zein gene, the oat glutelin gene, the sorghum kasirin gene,the rye secalin gene). Other promoters include those described by Brounet al. (1998), Potenza et al. (2004), US 20070192902 and US 20030159173.In an embodiment, the seed specific promoter is preferentially expressedin defined parts of the seed such as the cotyledon(s) or the endosperm.Examples of cotyledon specific promoters include, but are not limitedto, the FP1 promoter (Ellerstrom et al., 1996), the pea legumin promoter(Perrin et al., 2000), and the bean phytohemagglutnin promoter (Perrinet al., 2000). Examples of endosperm specific promoters include, but arenot limited to, the maize zein-1 promoter (Chikwamba et al., 2003), therice glutelin-1 promoter (Yang et al., 2003), the barley D-hordeinpromoter (Horvath et al., 2000) and wheat HMW glutenin promoters(Alvarez et al., 2000). In a further embodiment, the seed specificpromoter is not expressed, or is only expressed at a low level, in theembryo and/or after the seed germinates.

In another embodiment, the plant storage organ specific promoter is atuber specific promoter. Examples include, but are not limited to, thepotato patatin B33, PAT21 and GBSS promoters, as well as the sweetpotato sporamin promoter (for review, see Potenza et al., 2004). In apreferred embodiment, the promoter directs expression preferentially inthe pith of the tuber, relative to the outer layers (skin, bark) or theembryo of the tuber.

In another embodiment, the plant storage organ specific promoter is afruit specific promoter. Examples include, but are not limited to, thetomato polygalacturonase, E8 and Pds promoters, as well as the apple ACCoxidase promoter (for review, see Potenza et al., 2004). In a preferredembodiment, the promoter preferentially directs expression in the edibleparts of the fruit, for example the pith of the fruit, relative to theskin of the fruit or the seeds within the fruit.

When there are multiple promoters present, each promoter mayindependently be the same or different.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of thepolynucleotide, or may be heterologous with respect to the coding regionof the enzyme to be produced, and can be specifically modified ifdesired so as to increase translation of mRNA. For a review ofoptimizing expression of transgenes, see Koziel et al. (1996). The 5′non-translated regions can also be obtained from plant viral RNAs(Tobacco mosaic virus, Tobacco etch virus, Maize dwarf mosaic virus,Alfalfa mosaic virus, among others) from suitable eukaryotic genes,plant genes (wheat and maize chlorophyll a/b binding protein geneleader), or from a synthetic gene sequence. The present invention is notlimited to constructs wherein the non-translated region is derived fromthe 5′ non-translated sequence that accompanies the promoter sequence.The leader sequence could also be derived from an unrelated promoter orcoding sequence. Leader sequences useful in context of the presentinvention comprise the maize Hsp70 leader (U.S. Pat. Nos. 5,362,865 and5,859,347), and the TMV omega element.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the expression vector to thepolynucleotide of interest. The 3′ non-translated region of arecombinant DNA molecule contains a polyadenylation signal thatfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. The 3′ non-translated region can be obtained fromvarious genes that are expressed in, for example, plant cells. Thenopaline synthase 3′ untranslated region, the 3′ untranslated regionfrom pea small subunit Rubisco gene, the 3′ untranslated region fromsoybean 7S seed storage protein gene are commonly used in this capacity.The 3′ transcribed, non-translated regions containing the polyadenylatesignal of Agrobacterium tumor-inducing (Ti) plasmid genes are alsosuitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide by manipulating for example, the number ofcopies of the polynucleotide within a host cell, the efficiency withwhich those polynucleotide are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of polynucleotides defined herein include, butare not limited to, operatively linking the polynucleotide to ahigh-copy number plasmid, integration of the polynucleotide moleculeinto one or more host cell chromosomes, addition of vector stabilitysequences to the plasmid, substitutions or modifications oftranscription control signals (e.g., promoters, operators, enhancers),substitutions or modifications of translational control signals (e.g.,ribosome binding sites, Shine-Dalgarno sequences), modification of thepolynucleotide to correspond to the codon usage of the host cell, andthe deletion of sequences that destabilize transcripts.

In an embodiment, if the cell is a plant cell, the second exogenouspolynucleotide was introduced into the cell on a vector other than aviral vector.

Recombinant vectors may also contain: (a) one or more secretory signalswhich encode signal peptide sequences, to enable an expressedpolypeptide defined herein to be secreted from the cell that producesthe polypeptide, or which provide for localisation of the expressedpolypeptide, for example, for retention of the polypeptide in theendoplasmic reticulum (ER) in the cell, or transfer into a plastid,and/or (b) contain fusion sequences which lead to the expression ofnucleic acid molecules as fusion proteins. Examples of suitable signalsegments include any signal segment capable of directing the secretionor localisation of a polypeptide defined herein. Preferred signalsegments include, but are not limited to, Nicotiana nectarin signalpeptide (U.S. Pat. No. 5,939,288), tobacco extension signal, or the soyoleosin oil body binding protein signal. Recombinant vectors may alsoinclude intervening and/or untranslated sequences surrounding and/orwithin the nucleic acid sequence of a polynucleotide defined herein.

To facilitate identification of transformants, the recombinant vectordesirably comprises a selectable or screenable marker gene as, or inaddition to, the nucleic acid sequence of a polynucleotide definedherein. By “marker gene” is meant a gene that imparts a distinctphenotype to cells expressing the marker gene and thus, allows suchtransformed cells to be distinguished from cells that do not have themarker. A selectable marker gene confers a trait for which one can“select” based on resistance to a selective agent (e.g., a herbicide,antibiotic, radiation, heat, or other treatment damaging tountransformed cells). A screenable marker gene (or reporter gene)confers a trait that one can identify through observation or testing,that is, by “screening” (e.g., β-glucuronidase, luciferase, GFP or otherenzyme activity not present in untransformed cells). The marker gene andthe nucleotide sequence of interest do not have to be linked, sinceco-transformation of unlinked genes as for example, described in U.S.Pat. No. 4,399,216, is also an efficient process in for example, planttransformation. The actual choice of a marker is not crucial as long asit is functional (i.e., selective) in combination with the cells ofchoice such as a plant cell.

Exemplary selectable markers for selection of plant transformantsinclude, but are not limited to, a hyg gene which encodes hygromycin Bresistance; a neomycin phosphotransferase (nptII) gene conferringresistance to kanamycin, paromomycin, G418; a glutathione-S-transferasegene from rat liver conferring resistance to glutathione derivedherbicides as for example, described in EP 256223; a glutaminesynthetase gene conferring, upon overexpression, resistance to glutaminesynthetase inhibitors such as phosphinothricin as for example, describedin WO 87/05327; an acetyltransferase gene from Streptomycesviridochromogenes conferring resistance to the selective agentphosphinothricin as for example, described in EP 275957; a gene encodinga 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance toN-phosphonomethylglycine as for example, described by Hinchee et al.(1988); a bar gene conferring resistance against bialaphos as forexample, described in WO91/02071; a nitrilase gene such as bxn fromKlebsiella ozaenae which confers resistance to bromoxynil (Stalker etal., 1988); a dihydrofolate reductase (DHFR) gene conferring resistanceto methotrexate (Thillet et al., 1988); a mutant acetolactate synthasegene (ALS) which confers resistance to imidazolinone, sulfonylurea, orother ALS-inhibiting chemicals (EP 154,204); a mutated anthranilatesynthase gene that confers resistance to 5-methyl tryptophan; or adalapon dehalogenase gene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known; a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known; an aequorin gene(Prasher et al., 1985) which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof, or a luciferase (luc) gene (Ow etal., 1986) which allows for bioluminescence detection. By “reportermolecule” it is meant a molecule that, by its chemical nature, providesan analytically identifiable signal that facilitates determination ofpromoter activity by reference to protein product.

Preferably, the recombinant vector is stably incorporated into thegenome of the cell such as the plant cell. Accordingly, the recombinantvector may comprise appropriate elements which allow the vector to beincorporated into the genome, or into a chromosome of the cell.

Transfer Nucleic Acids

Transfer nucleic acids can be used to deliver an exogenouspolynucleotide to a cell and comprise one, preferably two, bordersequences and a polynucleotide of interest. The transfer nucleic acidmay or may not encode a selectable marker. Preferably, the transfernucleic acid forms part of a binary vector in a bacterium, where thebinary vector further comprises elements which allow replication of thevector in the bacterium, selection, or maintenance of bacterial cellscontaining the binary vector. Upon transfer to a eukaryotic cell, thetransfer nucleic acid component of the binary vector is capable ofintegration into the genome of the eukaryotic cell.

As used herein, the term “extrachromosomal transfer nucleic acid” refersto a nucleic acid molecule that is capable of being transferred from abacterium such as Agrobacterium sp., to a eukaryotic cell such as aplant leaf cell. An extrachromosomal transfer nucleic acid is a geneticelement that is well-known as an element capable of being transferred,with the subsequent integration of a nucleotide sequence containedwithin its borders into the genome of the recipient cell. In thisrespect, a transfer nucleic acid is flanked, typically, by two “border”sequences, although in some instances a single border at one end can beused and the second end of the transferred nucleic acid is generatedrandomly in the transfer process. A polynucleotide of interest istypically positioned between the left border-like sequence and the rightborder-like sequence of a transfer nucleic acid. The polynucleotidecontained within the transfer nucleic acid may be operably linked to avariety of different promoter and terminator regulatory elements thatfacilitate its expression, that is, transcription and/or translation ofthe polynucleotide. Transfer DNAs (T-DNAs) from Agrobacterium sp. suchas Agrobacterium tumefaciens or Agrobacterium rhizogenes, and man madevariants/mutants thereof are probably the best characterized examples oftransfer nucleic acids. Another example is P-DNA (“plant-DNA”) whichcomprises T-DNA border-like sequences from plants.

As used herein, “T-DNA” refers to for example, T-DNA of an Agrobacteriumtumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid,or man made variants thereof which function as T-DNA. The T-DNA maycomprise an entire T-DNA including both right and left border sequences,but need only comprise the minimal sequences required in cis fortransfer, that is, the right and T-DNA border sequence. The T-DNAs ofthe invention have inserted into them, anywhere between the right andleft border sequences (if present), the polynucleotide of interestflanked by target sites for a site-specific recombinase. The sequencesencoding factors required in trans for transfer of the T-DNA into aplant cell such as vir genes, may be inserted into the T-DNA, or may bepresent on the same replicon as the T-DNA, or preferably are in trans ona compatible replicon in the Agrobacterium host. Such “binary vectorsystems” are well known in the art.

As used herein, “P-DNA” refers to a transfer nucleic acid isolated froma plant genome, or man made variants/mutants thereof, and comprises ateach end, or at only one end, a T-DNA border-like sequence. Theborder-like sequence preferably shares at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90% or at least 95%, butless than 100% sequence identity, with a T-DNA border sequence from anAgrobacterium sp. such as Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Thus, P-DNAs can be used instead of T-DNAs to transfer anucleotide sequence contained within the P-DNA from, for exampleAgrobacterium, to another cell. The P-DNA, before insertion of theexogenous polynucleotide which is to be transferred, may be modified tofacilitate cloning and should preferably not encode any proteins. TheP-DNA is characterized in that it contains, at least a right bordersequence and preferably also a left border sequence.

As used herein, a “border” sequence of a transfer nucleic acid can beisolated from a selected organism such as a plant or bacterium, or be aman made variant/mutant thereof. The border sequence promotes andfacilitates the transfer of the polynucleotide to which it is linked andmay facilitate its integration in the recipient cell genome. In anembodiment, a border-sequence is between 5-100 base pairs (bp) inlength, 10-80 bp in length, 15-75 bp in length, 15-60 bp in length,15-50 bp in length, 15-40 bp in length, 15-30 bp in length, 16-30 bp inlength, 20-30 bp in length, 21-30 bp in length, 22-30 bp in length,23-30 bp in length, 24-30 bp in length, 25-30 bp in length, or 26-30 bpin length. Border sequences from T-DNA from Agrobacterium sp. are wellknown in the art and include those described in Lacroix et al. (2008),Tzfira and Citovsky (2006) and Glevin (2003).

Whilst traditionally only Agrobacterium sp. have been used to transfergenes to plants cells, there are now a large number of systems whichhave been identified/developed which act in a similar manner toAgrobacterium sp. Several non-Agrobacterium species have recently beengenetically modified to be competent for gene transfer (Chung et al.,2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234,Sinorhizobium meliloti and Mezorhizobium loti. The bacteria are madecompetent for gene transfer by providing the bacteria with the machineryneeded for the transformation process, that is, a set of virulence genesencoded by an Agrobacterium Ti-plasmid and the T-DNA segment residing ona separate, small binary plasmid. Bacteria engineered in this way arecapable of transforming different plant tissues (leaf disks, calli andoval tissue), monocots or dicots, and various different plant species(e.g., tobacco, rice).

Direct transfer of eukaryotic expression plasmids from bacteria toeukaryotic hosts was first achieved several decades ago by the fusion ofmammalian cells and protoplasts of plasmid-carrying Escherichia coli(Schaffner, 1980). Since then, the number of bacteria capable ofdelivering genes into mammalian cells has steadily increased (Weiss,2003), being discovered by four groups independently (Sizemore et al.1995; Courvalin et al., 1995; Powell et al., 1996; Darji et al., 1997).

Attenuated Shigella flexneri, Salmonella typhimurium or E. coli that hadbeen rendered invasive by the virulence plasmid (pWR100) of S. flexnerihave been shown to be able to transfer expression plasmids afterinvasion of host cells and intracellular death due to metabolicattenuation. Mucosal application, either nasally or orally, of suchrecombinant Shigella or Salmonella induced immune responses against theantigen that was encoded by the expression plasmids. In the meantime,the list of bacteria that was shown to be able to transfer expressionplasmids to mammalian host cells in vitro and in vivo has been more thendoubled and has been documented for S. typhi, S. choleraesuis, Listeriamonocytogenes, Yersinia pseudotuberculosis, and Y. enterocolitica(Fennelly et al., 1999; Shiau et al., 2001; Dietrich et al., 1998; Henseet al., 2001; Al-Mariri et al., 2002).

In general, it could be assumed that all bacteria that are able to enterthe cytosol of the host cell (like S. flexneri or L. monocytogenes) andlyse within this cellular compartment, should be able to transfer DNA.This is known as ‘abortive’ or ‘suicidal’ invasion as the bacteria haveto lyse for the DNA transfer to occur (Grillot-Courvalin et al., 1999).In addition, even many of the bacteria that remain in the phagocyticvacuole (like S. typhimurium) may also be able to do so. Thus,recombinant laboratory strains of E. coli that have been engineered tobe invasive but are unable of phagosomal escape, could deliver theirplasmid load to the nucleus of the infected mammalian cell nevertheless(Grillot-Courvalin et al., 1998). Furthermore, Agrobacterium tumefacienshas recently also been shown to introduce transgenes into mammaliancells (Kunik et al., 2001).

As used herein, the terms “transfection”, “transformation” andvariations thereof are generally used interchangeably. “Transfected” or“transformed” cells may have been manipulated to introduce thepolynucleotide(s) of interest, or may be progeny cells derivedtherefrom.

Recombinant Cells

The invention also provides a recombinant eukaryotic cell, for example,a recombinant plant cell, animal cell or fungal cell, which is a hostcell transformed with one or more polynucleotides or vectors definedherein, or combination thereof. The term “recombinant cell” is usedinterchangeably with the term “transgenic cell” herein.

Suitable cells of the invention include any cell that can be transformedwith a polynucleotide or recombinant vector as defined herein, encodingfor example, a polypeptide or dsRNA described herein. The recombinantcell may be a cell in culture, a cell in vitro, or in an organism suchas for example, a plant, or in an organ such as, for example, a seed ora leaf. In an embodiment, the eukaryotic cell is a non-human cell.

Host cells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid. Host cells of the present invention either can beendogenously (i.e., naturally) capable of producing polypeptide(s)defined herein, in which case the recombinant cell derived therefrom hasan enhanced capability of producing the polypeptide(s), or can becapable of producing said polypeptide(s) only after being transformedwith at least one polynucleotide of the invention.

Host cells of the present invention can be any cell capable of producingat least one protein described herein, and include fungal (includingyeast), parasite, arthropod, animal, and plant cells. Preferred hostcells are yeast, animal and plant cells. In a preferred embodiment, theplant cell is a seed cell, in particular, a cell in a cotyledon orendosperm of a seed. In one embodiment, the cell is an animal cell. Theanimal cell may be of any type of animal such as, for example, anon-human animal cell, a non-human vertebrate cell, a non-humanmammalian cell, or cells of aquatic animals such as fish or crustacea,invertebrates, insects, etc. Non limiting examples of arthropod cellsinclude insect cells such as Spodoptera frugiperda (Sf) cells, forexample, Sf9, Sf21, Trichoplusia ni cells, and Drosophila S2 cells.

The host cells may be of an organism suitable for a fermentationprocess, such as, for example, Yarrowia lipolytica or other yeasts.

Transgenic Plants

The invention also provides a plant comprising exogenous polynucleotidesas defined herein, a cell of the invention, a DNA construct of theinvention, a vector of the invention, or a combination thereof. The term“plant” refers to whole plants, whilst the term “part thereof” refers toplant organs (e.g., leaves, stems, roots, flowers, fruit), single cells(e.g., pollen), seed, seed parts such as an embryo, endosperm, scutellumor seed coat, plant tissue such as vascular tissue, plant cells andprogeny of the same. As used herein, plant parts comprise plant cells.

As used herein, the term “plant” is used in it broadest sense. Itincludes, but is not limited to, any species of grass, ornamental ordecorative plant, crop or cereal (e.g., oilseed, maize, soybean), fodderor forage, fruit or vegetable plant, herb plant, woody plant, flowerplant, or tree. It is not meant to limit a plant to any particularstructure. It also refers to a unicellular plant (e.g., microalga). Theterm “part thereof” in reference to a plant refers to a plant cell andprogeny of same, a plurality of plant cells that are largelydifferentiated into a colony (e.g., volvox), a structure that is presentat any stage of a plant's development, or a plant tissue. Suchstructures include, but are not limited to, leaves, stems, flowers,fruits, nuts, roots, seed, seed coat, embryos. The term “plant tissue”includes differentiated and undifferentiated tissues of plants includingthose present in leaves, stems, flowers, fruits, nuts, roots, seed, forexample, embryonic tissue, endosperm, dermal tissue (e.g., epidermis,periderm), vascular tissue (e.g., xylem, phloem), or ground tissue(comprising parenchyma, collenchyma, and/or sclerenchyma cells), as wellas cells in culture (e.g., single cells, protoplasts, callus, embryos,etc.). Plant tissue may be in planta, in organ culture, tissue culture,or cell culture.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a transgene not found in a wild-typeplant of the same species, variety or cultivar. Transgenic plants asdefined in the context of the present invention include plants and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide definedherein in the desired plant or part thereof. The term “transgenic plantparts” has a corresponding meaning.

The terms “seed” and “grain” are used interchangeably herein. “Grain”refers to mature grain such as harvested grain or grain which is stillon a plant but ready for harvesting, but can also refer to grain afterimbibition or germination, according to the context. Mature graincommonly has a moisture content of less than about 18-20%. “Developingseed” as used herein refers to a seed prior to maturity, typically foundin the reproductive structures of the plant after fertilisation oranthesis, but can also refer to such seeds prior to maturity which areisolated from a plant.

As used herein, the term “vegetative tissue” or “vegetative plant part”or variants thereof is any plant tissue, organ or part that does notinclude the organs for sexual reproduction of plants or the seed bearingorgans or the closely associated tissues or organs such as flowers,fruits and seeds. Vegetative tissues and parts include at least plantleaves, stems (including bolts and tillers but excluding the heads),tubers and roots, but excludes flowers, pollen, seed including the seedcoat, embryo and endosperm, fruit including mesocarp tissue,seed-bearing pods and seed-bearing heads. In one embodiment, thevegetative part of the plant is an aerial plant part. In another orfurther embodiment, the vegetative plant part is a green part such as aleaf or stem. Vegetative parts include those parts principally involvedin providing or supporting the photosynthetic capacity of the plant orrelated function, or anchoring the plant.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to store energy in the form of for example, proteins,carbohydrates, lipid. Examples of plant storage organs are seed, fruit,tuberous roots, and tubers. A preferred plant storage organ of theinvention is seed.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes,tapioca, rice, sorghum, millet, cassava, barley, or pea), or otherlegumes. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruits. The plants may be vegetable orornamental plants. The plants of the invention may be: corn (Zea mays),canola (Brassica napus, Brassica rapa ssp.), other Brassicas such as,for example, rutabaga (Brassica napobrassica), mustard (Brassicajuncea), Ethiopian mustard (Brassica carinata), crambe (Crambeabyssinica), camelina (Camelina sativa), sugarbeet (Beta vulgaris),clover (Trifolium sp.), flax (Linum usitatissimum), alfalfa (Medicagosaliva), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghumbicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritiumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumhirsutum), sweet potato (Lopmoea batatus), cassava (Manihot esculenta),coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananacomosus), citris tree (Citrus spp.), cocoa (Theobroma cacao), tea(Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig(Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive(Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia intergrifolia), almond (Prunusamygdalus), jatropha (Jatropha curcas), lupins, Eucalypts, palm, nutsage, pongamia, oats, or barley.

Other preferred plants include C4 grasses such as Andropogon gerardi,Bouteloua curtipendula, B. gracilis, Buchloe dactyloides, Panicumvirgatum, Schizachyrium scoparium, Miscanthus species for example,Miscanthus x giganteus and Miscanthus sinensis, Sorghastrum nutans,Sporobolus cryptandrus, Switchgrass (Panicum virgatum), sugarcane(Saccharum oficinarum), Brachyaria; C3 grasses such as Elymuscanadensis, the legumes Lespedeza capitata and Petalostemum villosum,the forb Aster azureus; and woody plants such as Quercus ellipsoidalisand Q. macrocarpa.

In a preferred embodiment, the plant is an angiosperm.

In an embodiment, the plant is an oilseed plant, preferably an oilseedcrop plant. As used herein, an “oilseed plant” is a plant species usedfor the commercial production of lipid from the seeds of the plant. Theoilseed plant may be oil-seed rape (such as canola), maize, sunflower,safflower, soybean, sorghum, flax (linseed) or sugar beet. Furthermore,the oilseed plant may be other Brassicas, cotton, peanut, poppy,rutabaga, mustard, castor bean, sesame, safflower, or nut producingplants. The plant may produce high levels of lipid in its fruit such asolive, oil palm or coconut. Horticultural plants to which the presentinvention may be applied are lettuce, endive, or vegetable Brassicasincluding cabbage, broccoli, or cauliflower. The present invention maybe applied in tobacco, cucurbits, carrot, strawberry, tomato, or pepper.

In a preferred embodiment, the transgenic plant is homozygous for eachand every gene that has been introduced (transgene) so that its progenydo not segregate for the desired phenotype. The transgenic plant mayalso be heterozygous for the introduced transgene(s), preferablyuniformly heterozygous for the transgene such as for example, in F1progeny which have been grown from hybrid seed. Such plants may provideadvantages such as hybrid vigour, well known in the art.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and Christou and Klee, Handbook of Plant Biotechnology,John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”“integrated” and variations thereof refer to the integration of thepolynucleotide into the genome of the cell such that they aretransferred to progeny cells during cell division without the need forpositively selecting for their presence. Stable transformants, orprogeny thereof, can be selected by any means known in the art such asSouthern blots on chromosomal DNA, or in situ hybridization of genomicDNA.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues, plant organs, or explants in tissueculture, for either transient expression, or for stable integration ofthe DNA in the plant cell genome. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see for example, U.S. Pat. Nos. 5,177,010, 5,104,310,5,004,863, or U.S. Pat. No. 5,159,135). The region of DNA to betransferred is defined by the border sequences, and the intervening DNA(T-DNA) is usually inserted into the plant genome. Further, theintegration of the T-DNA is a relatively precise process resulting infew rearrangements. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.Preferred Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985)).

Acceleration methods that may be used include for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like. A particular advantage ofmicroprojectile bombardment, in addition to it being an effective meansof reproducibly transforming monocots, is that neither the isolation ofprotoplasts, nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intoZea mays cells by acceleration is a biolistics α-particle deliverysystem, that can be used to propel particles coated with DNA through ascreen such as a stainless steel or Nytex screen, onto a filter surfacecovered with corn cells cultured in suspension. A particle deliverysystem suitable for use with the present invention is the heliumacceleration PDS-1000/He gun available from Bio-Rad Laboratories.

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein, one may obtain up to 1000 or more foci ofcells transiently expressing a marker gene. The number of cells in afocus that express the gene product 48 hours post-bombardment oftenrange from one to ten and average one to three.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includeparticle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (U.S. Pat. Nos. 5,451,513, 5,545,818, 5,877,402,5,932,479, and WO 99/05265).

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions that influence the physiological state of therecipient cells and that may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage, or cell cycle of the recipientcells, may be adjusted for optimum transformation. The execution ofother routine adjustments will be known to those of skill in the art inlight of the present disclosure.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).

Other methods of cell transformation can also be used and include butare not limited to the introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988)). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolynucleotide is cultivated using methods well known to one skilled inthe art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908), soybean (U.S.Pat. Nos. 5,569,834, 5,416,011), Brassica (U.S. Pat. No. 5,463,174),peanut (Cheng et al., 1996), and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447,PCT/US97/10621, U.S. Pat. Nos. 5,589,617, 6,541,257, and other methodsare set out in WO 99/14314. Preferably, transgenic wheat or barleyplants are produced by Agrobacterium tumefaciens mediated transformationprocedures. Vectors carrying the desired polynucleotide may beintroduced into regenerable wheat cells of tissue cultured plants orexplants, or suitable plant systems such as protoplasts.

The regenerable wheat cells are preferably from the scutellum ofimmature embryos, mature embryos, callus derived from these, or themeristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics.

A transgenic plant formed using Agrobacterium or other transformationmethods typically contain a single genetic locus on one chromosome. Suchtransgenic plants can be referred to as being hemizygous for the addedgene(s). More preferred is a transgenic plant that is homozygous for theadded gene(s), that is, a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by self-fertilisinga hemizygous transgenic plant, germinating some of the seed produced andanalyzing the resulting plants for the gene of interest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

Polypeptides

The terms “polypeptide” and “protein” are generally usedinterchangeably.

A polypeptide or class of polypeptides may be defined by the extent ofidentity (% identity) of its amino acid sequence to a reference aminoacid sequence, or by having a greater % identity to one reference aminoacid sequence than to another. The % identity of a polypeptide to areference amino acid sequence is typically determined by GAP analysis(Needleman and Wunsch, 1970; GCG program) with parameters of a gapcreation penalty=5, and a gap extension penalty=0.3. The query sequenceis at least 100 amino acids in length and the GAP analysis aligns thetwo sequences over a region of at least 100 amino acids. Even morepreferably, the query sequence is at least 250 amino acids in length andthe GAP analysis aligns the two sequences over a region of at least 250amino acids. Even more preferably, the GAP analysis aligns two sequencesover their entire length. The polypeptide or class of polypeptides mayhave the same enzymatic activity as, or a different activity than, orlack the activity of, the reference polypeptide. Preferably, thepolypeptide has an enzymatic activity of at least 10% of the activity ofthe reference polypeptide.

As used herein a “biologically active fragment” is a portion of apolypeptide defined herein which maintains a defined activity of afull-length reference polypeptide for example, silencing suppressoractivity. Biologically active fragments as used herein exclude thefull-length polypeptide. Biologically active fragments can be any sizeportion as long as they maintain the defined activity. Preferably, thebiologically active fragment maintains at least 10% of the activity ofthe full length polypeptide.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 40%, more preferably at least50%, more preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, more preferably at least 96%, more preferably at least97%, more preferably at least 98%, more preferably at least 99%, morepreferably at least 99.1%, more preferably at least 99.2%, morepreferably at least 99.3%, more preferably at least 99.4%, morepreferably at least 99.5%, more preferably at least 99.6%, morepreferably at least 99.7%, more preferably at least 99.8%, and even morepreferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides defined herein can beprepared by introducing appropriate nucleotide changes into a nucleicacid defined herein, or by in vitro synthesis of the desiredpolypeptide. Such mutants include for example, deletions, insertions, orsubstitutions of residues within the amino acid sequence. A combinationof deletions, insertions and substitutions can be made to arrive at thefinal construct, provided that the final polypeptide product possessesthe desired characteristics.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rationale designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess, for example, silencing suppressor activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries for example, by (1) substituting first with conservative aminoacid choices and then with more radical selections depending upon theresults achieved, (2) deleting the target residue, or (3) insertingother residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in thepolypeptide removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis include sitesidentified as the active site(s). Other sites of interest are those inwhich particular residues obtained from various strains or species areidentical. These positions may be important for biological activity.These sites, especially those falling within a sequence of at leastthree other identically conserved sites, are preferably substituted in arelatively conservative manner. Such conservative substitutions areshown in Table 1 under the heading of“exemplary substitutions”.

In a preferred embodiment a mutant/variant polypeptide has only, or notmore than, one or two or three or four conservative amino acid changeswhen compared to a naturally occurring polypeptide. Details ofconservative amino acid changes are provided in Table 1. As the skilledperson would be aware, such minor changes can reasonably be predictednot to alter the activity of the polypeptide when expressed in arecombinant cell. Furthermore, the skilled person can easily alignedrelated molecules, such as the V2-like proteins provided as SEQ ID NOs 1and 38 to 51, to identify suitable variants based on conserved andnon-conserved amino acids.

TABLE 1 Exemplary substitutions. Original Exemplary ResidueSubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gin; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gin Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,alaDirected Evolution

In directed evolution, random mutagenesis is applied to a protein, and aselection regime is used to pick out variants that have the desiredqualities, for example, increased silencing suppressor activity. Furtherrounds of mutation and selection are then applied. A typical directedevolution strategy involves three steps:

1) Diversification: The gene encoding the protein of interest is mutatedand/or recombined at random to create a large library of gene variants.Variant gene libraries can be constructed through error prone PCR (see,for example, Cadwell and Joyce, 1992), from pools of DNaseI digestedfragments prepared from parental templates (Stemmer, 1994a; Stemmer,1994b; Crameri et al., 1998; Coco et al., 2001) from degenerateoligonucleotides (Ness et al., 2002, Coco, 2002) or from mixtures ofboth, or even from undigested parental templates (Zhao et al., 1998;Eggert et al., 2005; Jezequel et al., 2008) and are usually assembledthrough PCR. Libraries can also be made from parental sequencesrecombined in vivo or in vitro by either homologous or non-homologousrecombination (Ostermeier et al., 1999; Volkov et al., 1999; Sieber etal., 2001). Variant gene libraries can also be constructed bysub-cloning a gene of interest into a suitable vector, transforming thevector into a “mutator” strain such as the E. coli XL-1 red (Stratagene)and propagating the transformed bacteria for a suitable number ofgenerations. Variant gene libraries can also be constructed bysubjecting the gene of interest to DNA shuffling (i.e., in vitrohomologous recombination of pools of selected mutant genes by randomfragmentation and reassembly) as broadly described by Harayama (1998).

2) Selection: The library is tested for the presence of mutants(variants) possessing the desired property using a screen or selection.Screens enable the identification and isolation of high-performingmutants by hand, while selections automatically eliminate allnonfunctional mutants. A screen may involve screening for the presenceof known conserved amino acid motifs. Alternatively, or in addition, ascreen may involve expressing the mutated polynucleotide in a hostorganism or part thereof and assaying the level of activity andoptionally, expressing the parent (unmutated) polynucleotide.Alternatively, the screen may involve feeding the organism or partthereof labelled substrate and determining the level of substrate orproduct in the organism or part thereof relative to a correspondingorganism or part thereof lacking the mutated polynucleotide andoptionally, expressing the parent (unmutated) polynucleotide.

3) Amplification: The variants identified in the selection or screen arereplicated many fold, enabling researchers to sequence their DNA inorder to understand what mutations have occurred.

Together, these three steps are termed a “round” of directed evolution.Most experiments will entail more than one round. In these experiments,the “winners” of the previous round are diversified in the next round tocreate a new library. At the end of the experiment, all evolved proteinor polynucleotide mutants are characterized using biochemical methods.

Rational Design

A protein can be designed rationally, on the basis of known informationabout protein structure and folding. This can be accomplished by designfrom scratch (de novo design) or by redesign based on native scaffolds(see, for example, Hallinga, 1997; and Lu and Berry, Protein StructureDesign and Engineering, Handbook of Proteins 2, 1153-1157 (2007)).Protein design typically involves identifying sequences that fold into agiven or target structure and can be accomplished using computer models.Computational protein design algorithms search the sequence-conformationspace for sequences that are low in energy when folded to the targetstructure. Computational protein design algorithms use models of proteinenergetics to evaluate how mutations would affect a protein's structureand function. These energy functions typically include a combination ofmolecular mechanics, statistical (i.e. knowledge-based), and otherempirical terms. Suitable available software includes IPRO (InterativeProtein Redesign and Optimization), EGAD (A Genetic Algorithm forProtein Design), Rosetta Design, Sharpen, and Abalone.

Also included within the scope of the invention are polypeptides definedherein which are differentially modified during or after synthesis forexample, by biotinylation, benzylation, glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. These modifications may serve to increase thestability and/or bioactivity of the polypeptide.

Uses

The cells of the invention with an increased level of an RNA of interestand/or amount of protein encoded by the RNA of interest, and a reducedlevel of target RNA encoded by a first polynucleotide of interest and/oramount of the protein encoded by the target RNA, can have a wide rangeof desired properties which influence, for example, an agronomic trait,insect resistance, disease resistance, herbicide resistance, sterility,grain characteristics, and the like. The encoded RNAs may be involved inmetabolism of oil, starch, carbohydrates, nutrients, etc., or may beresponsible for the synthesis of proteins, peptides, fatty acids,lipids, waxes, oils, starches, sugars, carbohydrates, flavors, odors,toxins, carotenoids, hormones, polymers, flavonoids, storage proteins,phenolic acids, alkaloids, lignins, tannins, celluloses, glycoproteins,glycolipids, etc.

In a particular example, the plants produced increased levels of enzymesfor oil production in plants such as Brassicas, for example oilseed rapeor sunflower, safflower, flax, cotton, soybean or maize; enzymesinvolved in starch synthesis in plants such as potato, maize, andcereals such as wheat barley or rice; enzymes which synthesize, orproteins which are themselves, natural medicaments, such aspharmaceuticals or veterinary products.

Types of polypeptides that are contemplated for production in a cell ofthe present invention include pharmaceutical proteins for use inmammals, including man, such as insulin, preproinsulin, proinsulin,glucagon, interferons such as α-interferon and γ-interferon,blood-clotting factors such as Factor VII, VIII, IX, X, XI, and XII,fertility hormones such as luteinising hormone, follicle stimulatinghormone growth factors such as epidermal growth factor, platelet-derivedgrowth factor, granulocyte colony stimulating factor, prolactin,oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone,calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO),enzymes such as β-glucocerebrosidase, haemoglobin, serum albumin,collagen, growth hormone, human serum albumin, human-secreted alkalinephosphatase, aprotinin, al-antitrypsin, IgG1 (phosphonate ester), IgM(neuropeptide hapten), SIgA/G (Streptococcus mutans adhesin),scFv-bryodin 1 immunotoxin (CD 40), IgG (HSV), LSC (HSV) and the like.

Furthermore, the cells of the invention can be used for the productionof specific antibodies, including antibody-related molecules or activefragments thereof which bind, for example, bone morphogenetic proteinreceptor-type IB; E16; STEAP1; MPF; Napi3b; Sema 5b; PSCA; Endothelintype B receptor; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2;HER2; NCA; MDP; IL20Rα; Brevican; EphB2R; ASLG659; PSCA; GEDA; Bcell-activating factor receptor; CD22; CD79a; CXCR5; HLA-DOB; P2X5;CD72; LY64; FcRH1; IRTA2; TENB2; CD20; VEGF including VEGF_A, B, C or D;p53; EGFR progesterone receptor; cathepsin D; Bcl-2; E cadherin; CEA;Lewis X; Ki67; PCNA; CD3; CD4; CD5; CD7; CD11c; CD11d; c-Myc; tau;PrPSC; or Aβ.

In addition, the cells of the invention can be used for the productionof an antigen, which may or may not be delivered by consumption of thestorage organ, examples of which include Hepatitis B virus envelopeprotein, rabies virus glycoprotein, Escherichia coli heat-labileentertoxin, Norwalk virus capsid protein, diabetes autoantigen, choleratoxin B subunit, cholera toxin B and A2 subunits, rotavirus entertoxinand enterotoxigenic E. coli fimbrial antigen fusions, porcinetransmissible gastroenteritis virus glycoprotein S, human rhinovirus 15(HRV-14) and human immunodeficiency virus type (HIV-1) epitopes, MinkEnteritis Virus epitopes, foot and mouth disease virus VP1 structuralprotein, human cytomegalovirus glycoprotein B, dental caries (S. mutans)antigens, and respiratory syncytial virus antigens.

In an embodiment, the target RNA encodes a polypeptide other than aprotein having sn-2 glycerol-3-phosphate acyltransferase (GPAT) activityand/or the RNA of interest encodes a polypeptide other than a proteinhaving monoacylglycerol acyltransferase (MGAT) activity. In anembodiment, the eukaryotic cell is a cell other than an Arabidopsisthaliana cell.

EXAMPLES Example 1. General Materials and Methods

Expression of Genes in Plant Cells in a Transient Expression System

Genes were expressed in plant cells using a transient expression systemessentially as described by Voinnet et al. (2003) and Wood et al.(2009). Chimeric binary vectors, 35S:p19 and 35S:V2, for expression ofthe p19 and V2 viral silencing suppressors, respectively, wereseparately introduced into Agrobacterium tumefaciens strain GV3101:mp90.All other binary vectors containing a coding region to be expressed by apromoter, such as the strong constitutive CaMV 35S promoter, wereintroduced into Agrobacterium tumefaciens strain AGL1. The recombinantcells were grown to stationary phase at 28° C. in LB broth supplementedwith 50 mg/L rifampicin and either 50 mg/L kanamycin or 80 mg/Lspectinomycin according to the selectable marker gene on the binaryvector. Acetosyringone (100 μM) was added to the bacterial cultures andgrowth continued a further 2 hours for the induction of virulencefactors. The bacteria were pelleted by centrifugation at 3000 g for 5min at room temperature before being resuspended to OD600=2.0 ininfiltration buffer containing 10 mM MES pH 5.7, 10 mM MgCl₂ and 100 μMacetosyringone. The cells were then incubated at 28° C. with shaking foranother 30 minutes and a volume of each culture required to reach afinal concentration of OD600=0.3 added to a fresh tube. Mixed culturescomprising genes to be expressed included either of the 35S:p19 or35S:V2 constructs in Agrobacterium unless otherwise stated. The finalvolume was made up with the infiltration buffer.

Leaves were then infiltrated with the culture mixture and the plantswere typically grown for a further three to five days after infiltrationbefore leaf discs were recovered for total lipid isolation. Time coursesof GFP expression were conducted on the intact leaves from the first dayafter infiltration through to 7 days post-infiltration (dpi). N.benthamiana plants were grown in growth cabinets under a constant 24° C.with a 14/10 light/dark cycle with a light intensity of approximately200 lux using Osram ‘Soft White’ fluorescent lighting placed directlyover plants. Typically, 6 week old plants were used for experiments andtrue leaves that were nearly fully-expanded were infiltrated. Allnon-infiltrated leaves were removed by post infiltration to avoidshading.

Lipid Analysis

Total Lipid Isolation and Fractionation

Tissue samples were freeze-dried, weighed and total lipids extractedfrom samples of approximately 30 mg dry weight as described by Bligh andDyer (1959). When required, TAG fractions were separated from otherlipid components using a 2-phase thin-layer chromatography (TLC) systemon pre-coated silica gel plates (Silica gel 60, Merck). An extractedlipid sample equivalent to 10 mg dry weight of leaf tissue waschromatographed in a first phase with hexane/diethyl ether (98/2 v/v) toremove non-polar waxes and then in a second phase using hexane/diethylether/acetic acid (70/30/1 v/v/v). When required, polar lipids wereseparated from non-polar lipids in lipid samples extracted from anequivalent of 5 mg dry weight of leaves using two-dimensional TLC(Silica gel 60, Merck), using chloroform/methanol/water (65/25/4 v/v/v)for the first direction and chloroform/methanol/NHOH/ethylpropylamine(130/70/10/1 v/v/v/v) for the second direction. The lipid spots, andappropriate standards run on the same TLC plates, were visualized bybrief exposure to iodine vapour, collected into vials andtransmethylated to produce FAME for GC analysis as follows.

Conversion of Fatty Acids to FAMEs

For total lipid analysis, with the exception of the analysis of DHScontent, lipid extracted from an equivalent of 10 mg of dry weight leafmaterial was transmethylated using a solution ofmethanol/HCl/dichloromethane (10/1/1 v/v/v) at 80° C. for 2 hr toproduce fatty acid methyl esters (FAME). For analysis of DHS in leaves,samples were transmethylated using the same reagents but with milderconditions, namely for 10 mins at 50° C., using DHS (Larodan Chemicals)as a calibration standard. The FAME were extracted into hexane,concentrated to near dryness under a stream of N₂ gas and quicklyreconstituted in hexane prior to analysis by GC.

DHS and eDHS were determined in total lipid samples by the followingmethod. Samples were directly treated with 0.1M sodium methoxide inmethanol/chloroform (10:1) in a sealed test tube with heating at 90° C.for 60 mins to convert lipids to FAMEs. When cool, the solution wasslightly acidified to pH 6-7 with acetic acid. Saline andhexane/chloroform (4:1 v/v) were added with vigorous shaking, and thehexane/chloroform layer containing FAMEs was transferred to a vial foranalysis.

Capillary Gas-Liquid Chromatography (GC)

FAMEs were analysed by gas chromatography (GC) using an AgilentTechnologies 6890N gas chromatograph (Palo Alto, Calif., USA) equippedwith an Equity™-1 fused silica capillary column (15 m×0.1 mm i.d., 0.1μm film thickness), an FID, a split/splitless injector and an AgilentTechnologies 7683 Series auto sampler and injector. Helium was used asthe carrier gas. Samples were injected in splitless mode at an oventemperature of 120° C. After injection, the oven temperature was raisedto 201° C. at 10° C.min⁻¹ and then to 270° C. at 5° C.min⁻¹ and held for20 min. Peaks were quantified with Agilent Technologies ChemStationsoftware (Rev B.03.01 (317), Palo Alto, Calif., USA). Peak responseswere similar for the fatty acids of authentic Nu-Check GLC standard-411(Nu-Check Prep Inc, MN, USA) which contained equal proportions of 31different fatty acid methyl esters, including 18:1, 18:0, 20:0 and 22:0.Slight variations of peak responses among peaks were balanced bymultiplying the peak areas by normalization factors of each peak. Theproportion of each fatty acid in total fatty acids of samples wascalculated on the basis of individual and total peaks areas for thefatty acids.

Analysis of FAMEs by Gas Chromatography-Mass Spectrometry

Analysis of FAMEs by gas chromatography-mass spectrometry (GCMS) wasconducted using a Varian 3800 equipped with a BPX70 capillary column(length 30 m, i.d. 0.32 mm, film thickness 0.25 μm, Phenomenex).Injections were made in the split mode using helium as the carrier gasand an initial column temperature of 60° C. raised at 20° C.min⁻¹ until180° C., then raised at 2.5° C.min⁻¹ until 190° C., then raised at 25°C.min⁻¹ until 260° C. and held for 2.2 min. Mass spectra were acquiredunder positive electron impact in full scan mode between 40-400 amu atthe rate of 2 scans per second using a Varian 1200 Single Quadrupolemass spectrometer. The mass spectra corresponding to each peak in thechromatogram was automatically compared with spectra of pure standards.Test spectra that matched standard spectra with a high degree ofaccuracy and eluted at the same time as an authentic standard or elutedat a plausible retention time, were identified. FAMEs were quantified bypeak area integration using Varian software and assuming equivalent MSresponse factors on a weight basis.

Quantification of TAG Via Iatroscan

One μl of each leaf extract was loaded on one Chromarod-SII for TLC-FIDIatroscan™ (Mitsubishi Chemical Medience Corporation—Japan). TheChromarod rack was then transferred into an equilibrated developing tankcontaining 70 ml of a Hexane/CHCl₃/2-Propanol/Formic acid(85/10.716/0.567/0.0567 v/v/v/v) solvent system. After 30 min ofincubation, the Chromarod rack was then dried for 3 min at 100° C. andimmediately scanned on an Iatroscan MK-6s TLC-FID analyser (MitsubishiChemical Medience Corporation—Japan). Peak areas of DAGE internalstandard and TAG were integrated using SIC-480II integration software(Version:7.0-E SIC System instruments Co., LTD—Japan).

TAG quantification was carried out in two steps. First, DAGE was scannedin all samples to correct the extraction yields after which concentratedTAG samples were selected and diluted. Next, TAG was quantified indiluted samples with a second scan according to the external calibrationusing glyceryl trilinoleate as external standard (Sigma-Aldrich).

Transformation of Arabidopsis thaliana

Chimeric vectors comprising genes to be used to transform Arabidopsiswere introduced into A. tumefaciens strain AGL1 and cells from cultureof the transformed Agrobacterium used to treat A. thaliana (ecotypeColumbia) plants using the floral dip method for transformation (Cloughand Bent, 1998).

Example 2. V2 Protein Acts as a Silencing Suppressor in Transient Assays

Construction of chimeric genes for expression of silencing suppressorsp19 or V2

The p19 protein from Tomato Bushy Stunt Virus (TBSV) (SEQ ID NO: 2) andthe V2 protein from Tomato Yellow Leaf Roll Virus (TYLRV) (SEQ ID NO: 1)have been characterised as viral suppressor proteins (VSP), functioningas silencing suppressors (Voinnet et al., 2003; Glick et al., 2008). p19binds to 21 nucleotide long siRNAs before they guide Argonaute-guidedcleavage of homologous RNA (Ye et al., 2003). V2 is an another silencingsuppressor that disrupts the function of the plant protein SGS3, aprotein thought to be involved in the production of double stranded RNAintermediates from ssRNA substrates (Elmayan et al., 1998; Mourrain etal., 2000; Beclin et al., 2002) either by directly binding to SGS3(Glick et al., 2008) or by binding dsRNA intermediates that contain a5′overhang structure and competitively excluding SGS3 from binding theseintermediates (Fukunaga and Doudna, 2009).

A DNA sequence encoding p19 (SEQ ID NO: 4), based on the genome sequenceof the Tomato Bushy Stunt Virus (Hillman et al., 1989) was chemicallysynthesised, including an NcoI site spanning the translation start ATGcodon. The DNA sequence was amplified by PCR and inserted into thepENTR/D-TOPO vector (Invitrogen), producing a plasmid designated pCW087(pENTR-p19). Gateway LR clonase reactions were then used to introducethe p19 coding sequence into plant binary vectors under the control ofeither the CaMV35S promoter, generating a construct designated pCW195(35S-p19), or the truncated napin promoter FP1, generating pCW082(FP1-p19). In addition, the entire FP1-p19-ocs3′ expression cassettefrom pCW082 was PCR amplified with SacI flanking sites and ligated intopCW141, a plant expression vector having a FP1-GFP gene as ascreenable/selectable seed marker, thus generating a plasmid designatedpCW164 (FP1-p19 and FP1-GFP). The presence of the FP1-GFP gene allowedthe non-destructive identification and selection of transformed T1 seedsin mixed null/T1 populations that resulted from the dipping techniquesused to transform Arabidopsis.

A DNA sequence encoding V2 (SEQ ID NO: 3), based on the Tomato YellowLeaf Curl Virus genome sequence (Glick et al., 2008), was chemicallysynthesised, included flanking NotI and AscI restriction sites to allowdirect cloning into the pENTR/D-TOPO vector (Invitrogen), generating aplasmid designated pCW192 (pENTR-V2). Gateway LR clonase reactions wereused to introduce the V2 gene into plant binary vectors under thecontrol of the 35S promoter (pCW197; 35S-V2) or for seed-specificexpression under the control of the truncated napin promoter, FP1(pCW195; FP1-V2).

The vector pUQ214 described in Brosnan et al. (2007) and comprising a35S-GFP gene, was used as an example of a target gene, expressing GFPunder the control of the 35S promoter. This binary vector included akanamycin resistance marker gene that can be used for selection oftransformed cells in plants if desired.

Function of the Suppressors in Plant Cells

In order to confirm the function of the V2 and p19 proteins assuppressors of silencing and therefore increasing transgene expression,Agrobacterium cells containing either of the 35S-driven VSP constructswere co-infiltrated together with Agrobacterium cells containing pUQ214into Nicotiana benthamiana leaves as follows. Transformants ofAgrobacterium tumefaciens strains separately harbouring each binaryvector were grown overnight at 28° C. in LB broth supplemented withantibiotics (50 mg/L kanamycin or 80 mg/L spectinomycin, dependent onthe selectable marker gene used) and rifampicin. Turbid cultures weresupplemented with 100 μM acetosyringone and grown for a further 2 hours.Cultures were centrifuged (4000×g for 5 min at room temperature) toharvest the cells and the cell pellets gently resuspended ininfiltration buffer (5 mM MES, 5 mM MgSO₄, pH 5.7, 100 μMacetosyringone) to an optical density of about 2.0. Cell suspensions forinfiltration were prepared, combining different transformants asrequired, so that each Agrobacterium strain was present at an OD6w. of0.3. The cell suspensions were infiltrated into the underside offully-expanded leaves of 5-6 week old N. benthamiana plants using a 1 mLsyringe without a needle, using gentle pressure. By these means, thecell suspensions entered primarily through the stomates and infiltratedthe mesophyll cell layer of the leaves. Infiltrated areas of leaves,indicated by the water-soaked region and commonly 3 to 4 cm in diameter,were circled by a permanent marker. Plants were housed in a 24° C. plantgrowth room with 14:10 light:dark cycle, where the light intensity was400-500 pEinsteins.m².s′ at the leaf surface provided by overheadfluorescent lighting (Philips TLD 35S/865 ‘Cold Daylight’). Under theseconditions, the Agrobacteria efficiently transfered the T-DNAs into theN. benthamiana cells.

GFP expression in the leaves was measured daily from 1-7 days after theinfiltrations by measuring the fluoresence under UV light. GFP imageswere captured on a digital SLR (Nikon D60; 55-200 mm lens) using theNightSea fluorescent light and filter set (NightSea, Bedford, Mass.,USA). Infiltrated leaves were generally left on the plant and werephotographed every day from 2-7 days post infiltration, thereby atime-course of GFP expression could be determined for the same set ofinfiltrations. Representative fluorescence photographs are shown in FIG.1 .

The 35S:GFP construct introduced in the absence of a VSP produced arelatively low level of fluorescence, indicative of GFP expression,peaking after 2-3 days and reducing thereafter. In contrast, when theGFP construct was co-infiltrated with either the p19 or the V2suppressor constructs, both the intensity and duration of fluorescencewere greatly increased, extending to and maintained beyond more than 7days post infiltration. These observations indicated enhanced expressionof the 35S:GFP gene in the leaf assays in the presence of the VSPs, andconfirmed their function as potent suppressor proteins that inhibitedthe endogenous co-suppression pathways in the plant cells.

Measurement of GFP Expression by Western Blot Analysis

GFP expression was also analysed by Western blot using a GFP specificantibody as follows. 1 cm² leaf samples were removed from theinfiltrated zones and subjected to denaturing protein extraction,polyacrylamide gel electrophoresis (PAGE; 12% gel) and blotting to PVDFmembrane essentially as described (Helliwell et al., 2006). GFP proteinwas detected using an anti-GFP monoclonal antibody (1:10000 dilution,Clontech) and goat anti-mouse HRP (1:5000 dilution, Promega) accordingto the suppliers instructions. Coomassie blue staining of high molecularproteins remaining in gels after the transfer to PVDF membranes was usedto confirm equal protein loading between samples. Protein size wasdetermined using the Pre-Stained PageRuler Protein Ladder (MBI-FermentasP7711S).

The results of the Western blot analyses confirmed the fluorescencedata, confirming the function of both p19 and V2 as silencingsuppressors (FIG. 2 ).

Example 3. RNAi Gene Silencing can Occur Simultaneously with SilencingSuppression

Hairpin RNAi Constructs Targeting GFP

A binary construct pUQ218 (Brosnan et al., 2007), containing both a35S-GFP gene and a 35S-hairpin encoding region targeted against GFP andwithin the same T-DNA region, was used when experiments used both GFPexpression and simultaneous GFP silencing activities in the same cell.The hairpin RNA comprised the first 380 bp of the GFP coding sequence,corresponding to nucleotides 1 to 380 of Accession No. U43284. A hpGFPbinary construct without the 35S-GFP gene was generated by removing the35S-GFP component via a NheI-AvrII digestion/religation reaction,creating pCW445 (35S-hpGFP).

Co-Expression of Silencing Suppressors and Silencing Constructs withTransgene Expression

The VSPs, V2 and p19, were compared in combination with GFP expressionfrom the 35S-GFP gene and a hairpin targeting GFP (hpGFP) to silence the35S-GFP gene, using transient assays by infiltration of the genes fromAgrobacterium into N. benthamiana leaves. These were compared to controlinfiltrations without the hpGFP, into adjacent spots on the same leaf atthe same time, to determine expression levels in the absence of thehairpin RNA. FIG. 1 , panel B, shows representative photographs of thefluorescence observed from 2 to 7 days post infiltration. Thecombination of pCW195 (35S-p19) and pUQ218 (containing both GFP andhpGFP) resulted in high levels of GFP expression, indicating that p19effectively suppressed silencing by the hairpin RNA of the GFPtransgene. In contrast, combinations of V2, 35S-GFP and hpGFP resultedin a near-total silencing of GFP. Complete silencing of GFP was achievedwith hpGFP in the absence of any VSP.

Experiments using pUQ218 generated equivalent results for GFP expressioncompared to the combination of separate vectors pUQ214 (35S-GFP) andpCW557 (35S-hpGFP). This indicated that the hairpin RNA construct wasefficiently introduced into cells via Agrobacterium in the experimentsdescribed above, and that it was not necessary to link the target geneand the silencing gene on a single construct in the transient leafassays.

Western blots of GFP protein levels (FIG. 2 ) using a specific antibodyas in Example 2 confirmed that the co-introduction of p19 suppressed thesilencing activity of hpGFP, thereby allowing strong GFP expression. Incontrast, only a low level of GFP expression was detected when thecombination of V2, GFP and hpGFP was introduced. This great differencebetween p19 and V2 with respect to suppressing the function of a hairpinRNA indicated that V2 may allow strong over-expression of transgenessimultaneously with hairpin-based RNAi strategies in the same cell.

Example 4. Silencing of an Endogenous Gene in the Presence of SilencingSuppressors

In order to test whether an endogenous gene could be silencedsimultaneously with expression of a silencing suppressor, a hairpin RNAconstruct was designed and made which would silence a FAD2 gene in N.benthamiana plants (NbFAD2) (SEQ ID NO: 11). FAD2 is a membrane-boundenzyme located on the endoplasmic reticulum (ER) which desaturates 18:1esterified on phosphatidylcholine (18:1-PC) to form 18:2-PC. Activity ofFAD2 can readily be assayed by analysing the fatty acid composition oflipid in the plant tissues and determining the ratio of 18:1 (oleicacid) to 18:2 (linoleic acid) in the total fatty acid. FAD2 is active inleaves of N. benthamiana as in other plants, resulting in low levels of18:1-PC in the leaves. As 18:1-PC is an important metabolite for a rangeof alternative fatty acids metabolic pathways, a chimeric gene was madewhich included an inverted repeat of a 660 basepair region of NbFAD2(SEQ ID NO: 12), corresponding to central portion of the endogenous 1151bp transcript, to silence NbFAD2 as follows.

Construction of Hairpin Construct Targeting NbFAD2

A 660 bp fragment of NbFAD2 was generated by RT-PCR from leaf total RNAusing primers designed against conserved regions of a Nicotianum tabacumFAD2 sequence in the Solgenomics database (SGN-U427167), namely forwardprimer NbFAD2F1 5′-TCATTGCGCACGAATGTGGCCACCAT-3′ (+451 bp co-ordinates)(SEQ ID NO: 13) and reverse primer NbFAD2R1 5′-CGAGAACAGATGGTGCACGACG-3′(+1112 bp co-ordinates) (SEQ ID NO: 14). Total RNA was isolated fromyoung N. benthamiana leaves using a Trizol-based method (Invitrogen andassociated literature). A Platinum Taq One-Step RT-PCR reaction(Invitrogen) was performed using the cycling conditions of 50° C. (10min), 94° C. (2 min) and 30 cycles of 50° C. (30 s)/72° C. (60 s)/92° C.(30s) and a final 72° C. (2 min). The NbFAD2 gene fragment wassubsequently ligated into pENTR11 and recombined using standard Gatewayprocedures into the pHellsgate8 vector (Helliwell et al., 2002) togenerate the plasmid designated pFN033. This construct had an invertedrepeat of the 660 bp fragment under the control of the 35S promoter,thereby producing, upon transcription, a RNA hairpin directed againstNbFAD2, hereafter named hpNbFAD2.

hpNbFAD2 was transformed into Agrobacterium tumefaciens strain AGL1 andinfiltrated into N. benthamiana leaves in combination with Agrobacteriacontaining the 35S:V2 or 35S:p19 constructs. Five days postinfiltration, infiltrated zones from leaves were sampled, total lipidextracted and the PC fraction analysed. The fatty acid analysis of thePC fraction of leaves infiltrated with combinations of hpNbFAD2 and V2showed a substantial increase in the 18:1-PC content from 9% 18:1-PC to39% 18:1-PC (FIG. 3 ). These percentages were based on the observedamounts of 18:1, 18:2 and 18:3 found on the PC fraction and expressed asa percentage of the sum of these three fatty acids. In comparison, thecombination of p19 and hpNbFAD2 resulted in partial silencing of FAD2activity, reflected in an increase from 8% 18:1-PC to 25% 18:1-PC, aresult indicating that hpNbFAD2 could silence the endogenous FAD2 geneto a moderate extent in the presence of co-expression of p19. Previouswork has shown that leaf cells infiltrated with a combination ofAgrobacteria strains, each containing a separate vector, received atleast one or more copies of T-DNA from each vector (Wood et al., 2009).This gave us confidence that the great majority of cells in the leafassays described above had received and expressed both the hairpin andthe suppressor encoding genes.

The increase in 18:1-PC levels was reflected in a reduction in the18:2-PC content in the cells. In contrast, the 18:3-PC levels nearly thesame, presumably due to the large amount of 18:3 generated in theFAD2-independent pathways found in the chloroplasts of leaves.

To establish that the suppressor and hairpin constructs were introducedinto the same cells efficiently, constructs were also made and testedwhich co-located the genes within the same T-DNA constructs, thusgenerating single T-DNAs with 35S-p19+35S-hpNbFAD2 and 35S-V2+35S-NbFAD2gene combinations. The entire 35S-p19-ocs3′ region of pCW194 was PCRamplified using the primers including MluI flanking sites, (underlined)namely Forward primer 5′ aacacttcgacgaattaattccaatcccaca-3′ (SEQ ID NO:15) and the OCS'3 Reverse primer 5′-ACGCGTCTGCTGAGCCTCGACATGTT-3′ (SEQID NO: 16). The amplified fragment was ligated into the unique MluI sitewithin pFN033 to create pCW701, containing 35S-p19+35S-hpNbFAD2. Usingthe same primers, the entire 35S-V2-ocs3′ region of pCW197 was PCRamplified and this amplicon was ligated into the unique MluI site ofpFN033 to create pCW702, containing 35S-p19+35S-hpNbFAD2. These vectorshaving the suppressor and hairpin encoding genes located within the sameT-DNA region were transformed into Agrobacterium strain AGL1 andinfiltrated into N. benthamiana leaves as before. Leaf tissues weresampled 5 dpi and the PC lipid fractions analysed for the 18:1, 18:2 and18:3 levels. The results were indistinguishable compared to the resultsobtained using genes introduced on separate vectors, the inventorsconcluded that essentially all of the transformable leaf cells intransient leaf assays received at least one copy of each T-DNA in theinfiltration mixtures.

Simultaneous Silencing of One Gene while Overexpressing a Second Gene

To test whether additional genes could be over-expressed with the aid ofa silencing suppressor while silencing the endogenous FAD2 gene,additional constructs were made for over-expression of genes encodingDGAT1 and oleosin in plant cells. All plant cells possess active lipidpathways producing lipid classes such as DAG and acyl-CoA (Ohlrogge andBrowse, 1995), however the esterification of these substrates via DGATto produce TAG only occurs at significant levels in specialised organs,such as oilseeds and pollen. The ectopic expression of AtDGAT1 in leaveshas been shown to generate increased levels of oils (Bouvier-Nave etal., 2000). Previous studies have also shown that AtDGAT1 has somesubstrate specificity for 18:1 and its elongation product, 20:1 (Katavicet al., 1995). Oleosins are amphipathic proteins whose propertiesposition these proteins on oil/hydrophilic interfaces, thereby creatinga coating surrounding oil droplets and forming so called ‘oil bodies’ inoil-generating tissues (Tzen et al., 1992). ‘Oil bodies’ are considereda long term storage organelle as the oleosin layer protects the TAG fromcatabolic processes such as TAG lipases. Seeds of Arabidopsis mutantslacking a functional oleosin, ole1, have significantly reduced 18:1contents and this 18:1 content was restored upon ectopic expression ofan oleosin encoding gene from sesame (Scott et al., 2010).

Synthesis and Use of Constructs to Overexpress DGAT1 and Oleosin

The coding region of the AtDGAT1 gene (SEQ ID NO: 10) was cloned fromArabidopsis Col-0 mRNA collected from developing embryos using primersbased on the Accession No. NG_127503. The amplicon was cloned intopENTR11 (Invitrogen) and recombined via an LR clonase reaction into a35S binary expression vector to create 35S-AtDGAT1. The oleosinconstruct was used as described by Scott et al. (2010). This constructhad a 35S promoter driving an oleosin coding region (SEQ ID NO: 6)isolated from sesame, encoding the protein with the amino acid sequenceof Accession No AF091840 (SEQ ID NO: 5), generating the constructdesignated 35S-Oleosin.

Combinations of Agrobacterial strains separately containing vectors fortransfer of genes encoding DGAT1, oleosin and p19 or V2 and in additionhpNbFAD2 were tested in N. benthamiana leaves and the oil content andfatty acid composition in the infiltrated tissues were analysed. Leafsamples were removed 5 dpi and freeze dried overnight. Lipids wereextracted from samples of about 30 mg dry weight using the method ofBligh and Dyer (1959). TAGs in the extracted lipids were separated frompolar lipids using a 2-phase TLC system on pre-coated silica gel plates(Silica gel 60, Merck). A lipid sample equivalent to 10 mg dry weight ofleaf tissue was first run with hexane/diethyl ether (98/2 by vol.) toremove very non-polar waxes and a second phase was run usinghexane/diethyl ether/acetic acid (70/30/1 by vol.). The lipid spots, andappropriate standards, were visualized by brief exposures to iodinevapour, collected into vials and transmethylated to produce FAME for GCanalysis as described in Example 1. The data are shown in FIG. 4 .

Leaves infiltrated with the genes encoding V2 and both DGAT1 and Oleosinhad an approximately 5 to 6 fold increase in the TAG content. Moreover,there was a doubling of the 18:1 level calculated as a percentage of thetotal fatty acids in the TAG fraction, indicating that the combinationof these two genes in the presence of the silencing suppressor enhancedthe formation (synthesis and accumulation) of leaf oils with increasedlevels of oleic acid. The further addition of the silencing constructhpNbFAD2 increased the 18:1 level in the leaf oil to either 44% whenusing V2 or to 35% using p19 as the VSP. This assay configurationconfirmed that both V2 and p19 allowed over-expression of transgenes,e.g. encoding AtDGAT1 and Oleosin. Although both silencing suppressorsallowed effective simultaneous endogenous FAD2 silencing, use of V2provided a greater extent of silencing than p19. From the efficiency ofthe 18:1 accumulation in TAGs, these observations were consistent withthe conclusion above that over-expression of the transgenes aided by theVSPs was occurring simultaneously in the same cells as the FAD2silencing.

In a further experiment to demonstrate that additional genes could beover-expressed with the aid of a silencing suppressor whilesimultaneously reducing expression of a second gene with a hairpin RNA,a construct was made to express a FAE1 enzyme (SEQ ID NO: 7). FAE1 is anenzyme that elongates saturated and monounsaturated fatty acidsesterified to CoA by adding 2 carbons to the acyl chain at the carboxylend of the fatty acid molecule (James et al., 1995). Previous studieshave shown that ectopic expression of AtFAE1 resulted in production of arange of new elongated fatty acids, including a series of so-calledvery-long chain fatty acids (VLCFA) due to the sequential activity ofAtFAE1 in cycles of elongation. The enzyme uses acyl-CoA substrates(Millar et al., 1998).

Synthesis of construct to express FAE1

The coding region of AtFAE1, TAIR Accession number 2139599, waschemically synthesised, subcloned into pGEMT-Easy and subcloned via theEcoRI flanking sites into the pENTR cloning vector, pCW306, to includethe AttL1 and AttL2 sites, to generate pCW327. A catalase-1 intron, fromthe castor bean catalase-1 gene, was ligated into the unique NotI sitejust upstream of the AtFAE1 ORF to generate pCW465, pENTR-intron-AtFAE1.LR clonase reactions were used to recombine the intron-AtFAE1 fragment(SEQ ID NO: 8) into a 35S expression vector, generating pCW483(35S-intron-AtFAE1). pCW483 was transformed into Agrobacterium strainAGL1 and transiently expressed in N. benthamiana leaves as above incombination with the other genes. A range of new elongation productswere found in leaves expressing AtFAE1, including a significant numberof VLCFA such as 20:1 (FIG. 11 ). Based on the known substratespecificity of AtFAE1, we reasoned that 18:1-CoA would be a preferredsubstrate for AtFAE1, however this substrate would only be foundwild-type leaves at low levels due to the activity of NbFAD2. Theinventors therefore combined the over-expression of AtFAE1 with hairpinbased silencing of NbFAD2 in the presence of the silencing suppressorV2.

These experiments demonstrated that silencing suppressors such as V2allowed over-expression of transgenes and the simultaneous silencing ofendogenous genes in the same cell, and allowed an optimised substratepool to be formed for metabolic engineering of fatty acids, e.g. 20:1and other VLCFA.

Example 5. Small RNA Analysis of Hairpin-Based Silencing of an Endogene

Hairpin-based RNAi constructs are known to generate populations of smallRNAs homologous to the hairpin, generally known as primary sRNAmolecules. These primary sRNAs can trigger the production of secondarysRNAs that are homologous to regions in the target RNA outside of thehairpin-targeted region. Such sRNAs are mostly 21, 22 or 24 nucleotidesin length, reflecting their biogenesis via a several pathways usingdifferent Dicer proteins. Each length may have specific functions intranscriptional gene silencing (TGS) and post-transcriptional genesilencing (PTGS). With the availability of deep sequencing technologies,the inventors investigated the small RNA populations arising fromhairpin-based gene silencing of the endogenous NbFAD2 gene by thehpNbFAD2 in the transient assays, as above.

Cloning of Full-Length Open-Reading Frame of the NbFAD2 Gene

First of all, the full length open reading frame of the FAD2 gene fromN. benthamiana was sequenced as follows. Genomic DNA was isolated from20 g fresh weight of N. benthamiana leaves using a method that reducedchloroplastic and mitochondrial DNA contamination (Peterson et al.,1997). High molecular weight DNA was randomly sheared into fragments ofapproximately 500 bp and ligated with TruSeq library adaptors togenerate a gDNA library. This library was sequenced on the HiSeq2000platform on a complete flowcell. High quality sequences were retained togenerate an alignment against the 660 bp hpNbFAD2 fragment (pFN033)using BowTie software. The full-length coding region of NbFAD2 wassubsequently cloned via high fidelity PCR using primers Forward5′-TTTATGGGAGCTGGTGGTAATATGT-3′ (SEQ ID NO: 17) and Reverse5′-CCCTCAGAATTTGTTrTGTACCAGAAA-3′ (SEQ ID NO: 18) (start and stop codonsunderlined) and sequence verified using BigDye3.1 sequencing techniques.

Small RNA Analysis

Deep sequencing methods were then used to analyse the populations ofsRNA generated from the hairpin RNAi silencing construct, hpNbFAD2, inleaves co-infiltrated with the construct encoding V2. Total RNA wasisolated from leaves 5 dpi using Trizol reagent (Invitrogen) accordingto the suppliers instructions. Small RNAs (15-40 nt size range) werepurified via gel electrophoresis and analysed on an Illumina GAxIImachine according to the manufacturers protocols.

Small RNAs having a sequence with identity to the NbFAD2 gene wereidentified and collated. The observed predominant sRNA size classes(20-24 nt) showed a non-uniform distribution across both the forward andreverse strands of the 660 bp target sequence (FIG. 8 ). Alignments ofthe small RNA reads against the full-length NbFAD2 open-reading framesequence indicated that all of the observed sRNAs with homology toNbFAD2 had identity with the region used to generate the hairpinconstruct, none to the non-targeted regions. Therefore, we concludedthat the combination of the V2 silencing suppressor and hpNbFAD2 did notgenerate secondary sRNAs at an observable frequency. The absolutenumbers of sRNA size classes showed that 20, 21, 22, 23 and 24 nt sRNArepresented 10%, 44%, 36%, 4% and 10% of all sRNA, respectively (FIG. 9). This result confirmed that hairpins generated primary sRNAs againstan endogenous gene and not secondary sRNAs, although we could notexclude an influence of the V2 suppressor in this result.

Example 6. Engineering a Transgenic Pathway for the Synthesis ofCyclopropanated Fatty Acids in Leaf Tissue

Oleic acid on the PC fraction is also the starting point for alternativemetabolic pathways, and therefore an alternative metabolic pathway whichuses oleic acid as a substrate was investigated as a system to comparedifferent VSP activities in transient leaf assays. Dihydrosterculic acid(DHS) was chosen as the desired product from oleic acid. DHS is acyclopropanated fatty acid that is produced by cyclopropane fatty acidsynthetases (CPFAS) using 18:1-PC as a substrate (FIG. 5 ). Twodifferent CPFAS genes were compared (FIG. 6 ) for their activity in leafassays to produce DHS, namely the Escherichia coli CPFAS (EcCPFAS) (SEQID NO: 24) and the C-terminal domain of the cotton CPFAS (SEQ ID NO:21), hereinafter termed GhCPFAS*, using leaf assays in combination withgenes encoding V2, hpNbFAD2, DGAT1 and Oleosin.

Construction of Genes to Over-Express EcCPFAS and GhCPFAS* for TransientExpression in Leaves and Seeds

A DNA sequence encoding an Escherichia coli CPFAS enzyme was chemicallysynthesised, based on Accession No. AE000261.1 from nucleotide 6129 fora length of 1143 bp (SEQ ID NO: 26). The encoded protein had the sameamino acid sequence as the E. coli protein, but the nucleotide sequencewas codon optimised with a codon bias more suited to eukaryoticexpression. The EcCPFAS-encoding fragment was cloned into the EcoRI siteof pCW391, generating pCW392, a binary T-DNA construct useful for leafassays (35S-EcCPFAS).

GhCPFAS*

The first plant CPFAS gene to be isolated and characterised inheterologous expression systems, namely SfCPFAS from Sterculia foetida,was found to possess a C-terminal portion of the enzyme with excellenthomology to known bacterial CPFAS enzymes and an N-terminal region withmotifs with homology to FAD-binding oxidases (Bao et al., 2002). A studyhas found that SfCPFAS is unusual and different to other plant fattyacid modifying enzymes by acting upon the 18:1 esterified to the sn1position of phosphatidylcholine (PC) (Bao et al., 2003).

The cotton CPFAS-1 gene shows some homology to the SfCPFAS gene and theexpression of full-length GhCPFAS-1 in tobacco BY2 cell cultureslikewise resulted in about 1% DHS (Yu et al., 2011). The expression offull-length GhCPFAS-1 in seeds of fad2 fae1 mutant backgrounds ofArabidopsis, having elevated levels of oleic acid in seeds, alsogenerated about 1% DHS (Yu et al., 2011). A comparison of thefull-length GhCPFAS to produce DHS and a protein truncated by the first409 amino acids, thus removing the FAD-binding oxidase domain, foundthat removal of the first 409 amino acids reduced DHS production inyeast by about 70% (Yu et al., 2011). Overall, these results indicatedthat plant CPFAS enzymes were capable of producing a low level of DHS intransgenic expression systems but that the first 409 amino acids wererequired for maximal activity. However, as described below the presentinventors were surprised to find that in plant cells the truncatedenzymes had enhanced CPFAS activity.

A DNA fragment encoding the C-terminal 469 amino acids of thefull-length GhCPFAS-1 enzyme, starting at nucleotide position 1248relative to the sequence in Accession No. AY574036 and using an internalin-frame ATG as the new start codon, was generated in RT-PCR reactionsusing total RNA isolated from cotton, to generate a nucleotide sequenceencoding (SEQ ID NO: 23) the modified protein GhCPFAS* (SEQ ID NO: 21).The predicted length of the protein was 469 amino acids and thereforeincluding only the region with homology to the bacterial CPFAS gene,without the N-terminal region having homology to FAD-binding oxidases.The PCR primers used to amplify this region of GhCPFAS-1 included SpeIflanking sites (underlined), and were Forward primer:5′-TTACTAGTATGGATGCTGCACATGGTATCT-3′ (SEQ ID NO: 19) and Reverse primer:5′-TTACTAGTTCAATCATCCATGAAGGAATATGCAGAA-3′ (SEQ ID NO: 20). The ampliconwas inserted into the SpeI site of 35S-pORE4 to generate pCW618(35S-GhCPFAS*).

The construct was introduced into Agrobacterium and used to infiltrateN. benthamiana leaves in transient assays as before, in variouscombinations with other genes. Analyses of the total lipid content ofthe infiltrated zones of these leaves indicated that GhCPFAS*efficiently produced DHS in leaves (FIG. 6 ). The level of DHS producedin the presence of GhCPFAS* was approximately 7% of the total fattyacids in leaf lipids, with an overall pathway conversion efficiency of47% for conversion of oleic acid to DHS. In comparison, EcCPFAS producedless than 1% DHS in total fatty acids in leaf lipids with a conversionefficiency of 4%. GhCPFAS* was therefore used throughout the remainderof this study.

In a further experiment, the production of DHS by GhCPFAS* was used todirectly compare the efficiency of p19 or V2 to aid the simultaneousover-expression of the GhCPFAS* transgene and silencing of the NbFAD2gene, that is, where silencing of an endogenous gene was required tomaximise flux into a novel biosynthetic pathway. Various combinations ofGhCPFAS*, DGAT1, Oleosin, V2, p19, and hpNbFAD2 were infiltrated into N.benthamiana leaves and the production of DHS determined (FIG. 7 ). Inthe absence of hpNbFAD2, a slightly greater level of DHS production wasobserved in the presence of p19 compared to V2. However, in the presenceof the hairpin hpNbFAD2, greater levels of DHS were observed with theuse of V2. V2 allowed the greatest levels of substrate (18:1) to beproduced and also the greatest levels of DHS production. Overall the useof V2 in the combined overexpression and silencing scenario generatedapproximately 30% more DHS in the leaf assays compared to the use ofp19.

A critical step in TAG synthesis pathways involves the removal of theacyl group from the PC head group into the CoA pool. Once acyl groupsenter the CoA pool, they become available for the TAG synthesis pathwaytermed the ‘Kennedy’ pathway that includes the last committed step ofTAG formation catalysed by the DGAT enzyme. The movement of DHS,produced on the PC fraction of leaves, into leaf TAGs was tested bycombining GhCPFAS* with DGAT1, Oleosin and hpNbFAD2 (FIG. 10 ). DHSproduced by GhCPFAS*, DGAT1 and Oleosin was found in leaf TAGs atapproximately 7% of the total fatty acid content in TAG, with aconversion efficiency of oleic acid to DHS of 55%. The inclusion ofhpNbFAD2 boosted the percentage of DHS in leaf TAG from 7% to 15%, whilethe conversion efficiency remained unchanged at 55%. These resultsindicated that the combination of V2 and hpNbFAD2 doubled the flux ofDHS into the metabolic pathway, using in additionCPFAS*+AtDGAT1+Oleosin, to produce plant oils having higherconcentrations of cyclopropanated fatty acids.

To demonstrate whether the DHS was exchanged readily between the PC andCoA pools, a further experiment was performed which added AtFAE1 to thecombination of enzymes. The inventors reasoned that the fatty acid DHS,containing a mid-chain propane ring, was likely to form a structuresimilar to and intermediate between that of a saturated and amonounsaturated C18 fatty acid and that if DHS was transferred from thePC fraction into the CoA pool, it would be a suitable substrate forAtFAE1 to produce elongated DHS (eDHS). To examine if DHS, produced onPC, was transferred into the CoA pool of leaves, the chimeric 35S:AtFAE1gene was included in combination with genes encoding V2, GhCPFAS* andhpNbFAD2, each under the control of the 35S promoter. The results of thefatty acid analysis are shown in FIG. 11 . Total lipids analysed 5 dpiwere enriched for DHS and a new metabolite. The new metabolite wasconfirmed as eDHS, an elongated product of DHS with an additional 2carbon atoms, by using standard GC/MS techniques (FIG. 12 ). Theconversion efficiency of DHS to eDHS averaged 15% across 6 samplescompared to the conversion of 18:1 to 20:1 which averaged 28%.Collectively, these experiments provided evidence that DHS produced onPC was moved efficiently into the CoA pool and accumulated into leafoils via expression of a combination of endogenous genes and transgenicgenes.

Example 7. Transgenic Plant Studies

EcCPFAS in Arabidopsis Seeds

The EcCPFAS fragment (Example 6) was cloned into the EcoRI site ofpCW442 generating pCW393 (FP1-EcCPFAS) a seed-specific expression vectorusing the truncated FP1 promoter to drive expression of EcCPFAS. Thispromoter is useful for expression of transgenes in oilseeds (Ellerstromet al., 1996). This vector was transformed into Agrobacteriumtumefaciens strain AGL1, and used to transform Arabidopsis plants of thefad2/fae1 double mutant background via the floral dip method. Transgenicseeds were selected on media containing kanamycin (40 mg/L) and T2 seedof these plants analysed for DHS content as described in Example 1.

Seven independent transformed lines of Arabidopsis were analysed and theDHS content ranged from trace levels through to 1% DHS, consistent withthe studies described above.

GhCPFAS in Seeds of Arabidopsis and Safflower

A plant binary expression vector was designed for the expression oftransgenes using a promoter derived from the promoter of the AtOlesoin1gene (TAIR website gene annotation At4g25140). The promoter was modifiedin that 6 basepairs within the 1192 bp sequence were omitted to deletecommon restriction enzyme sites. The AtOleosin promoter has been usedfor the strong seed-specific expression of transgenes in safflower andBrassica species (Nykiforuk et al., 2011; Van Rooijen and Moloney,1995). This promoter is thought to be bi-directional, directing not onlystrong seed-specific expression of transgenes placed at the 3′end of thepromoter, but also generating transcripts in the opposite direction fromthe 5′end of the promoter in a range of tissues. The Arabidopsis oleosinpromoter shares features of the Brassica napus promoter, characterisedto have a bi-functional nature (Sadanandom et al., 1996). The promoterwas chemically synthesised and subcloned into pGEMT-Easy and an EcoRIfragment of this vector was blunted via the Klenow enzyme fill-inreaction and ligated into the Klenow-blunted HindI site of pCW265(Belide et al., 2011), generating pCW600 (AtOleosinP::empty). ASpeI-flanked fragment of pCW618 encompassing the GhCPFAS* coding regionwas ligated into pCW600, generating pCW619 (AtOloesin:GhCPFAS*).

This pCW619 vector was introduced into Agrobacterium tumefaciens strainAGL1 and used to transform Arabidopsis of either the fad2 or fad2fae1mutant genotypes via the floral dip method. The same construct was alsoused to transform safflower of the variety S317 (high oleic background)via a method using grafting (Belide et al., 2011). 15 independenttransformed lines of the fad2 mutant of Arabidopsis transformed withpCW619 were obtained and T2 seeds of these plants are maturing. 20independent transformed lines of safflower S317 transformed with pCW619were generated and seeds of these plants are maturing. DHS contents inseeds are analysed and are elevated.

Discussion

These experiments showed that the silencing suppressor protein V2 wasadvantageous in allowing efficient over-expression of one or more genestogether with the silencing of genes, in the same cell. Although p19allowed excellent over-expression of transgenes and was more effectivethan V2 as a silencing suppressor, p19 also partially blockedhairpin-based silencing of endogenous genes. It is postulated hereinthat V2 and its functional homologs block the co-suppression pathwaywhich utilises RNA dependent RNA polymerase and SGS3 and therebymaximises expression of a desired gene, but has little effect on thehairpin-RNA or microRNA silencing pathways and thereby allowsconcomitant gene silencing. The use of V2 also allowed the efficientexpression of numerous additional genes to the cells to form a newmetabolic pathway, using either individual (separate) vectors or genescombined on single constructs, and thereby entire transgenic pathwayscould be assembled and tested within a few days in the transient assays.The inventors used the V2-based leaf assays to determine that GhCPFAS*was much better than EcCPFAS in producing DHS.

Finally, the optimised leaf assays demonstrated that the unusual fattyacid DHS, produced on PC, was efficiently unloaded into the CoA pool andaccumulated in leaf oil. The accumulation of 15% DHS in leaf oilsreported here with GhCPFAS* exceeds levels reported with any CPFASexpressed in any plant cell reported in previous studies. Such efficientmovements of DHS between lipid pools in leaf cells indicated that leavesmight be an ideal location for the production of DHS rather than oralternative to oilseeds.

Beyond oleochemical engineering, we envision that silencing suppressorssuch as V2 and its homologs will be useful for a range of basic andapplied areas of research. Transient leaf assays are currently beingdeveloped for the rapid production of personalised antibodies (Levy etal., 2008), however the plant glycosylation and silylation patterns needto be ‘humanised’ for full efficacy, which requires silencing of severalgenes in the plant cells. The use of transient leaf expression systemsas described above may provide rapid production of antibodies moresuitable for human therapies, or allow gene-replacement surveys to beperformed.

Example 8. Combining Silencing Suppressors and microRNAs

Effect of V2 or p19 on the Activity of an Artificial miRNA inStably-Transformed Plants

Artificial miRNA (amiRNA) constructs may be processed by eukaryoticsilencing pathways including in plants to generate a 21 nt long doublestranded RNA with 2 nt 3′ overhangs, from which a single RNA strand isloaded into Argonaut proteins to guide targeted silencing of a gene ofinterest whilst the second strand (passenger strand) is degraded (Schwabet al., 2006). The highly specific sRNA created in amiRNA approaches canbe contrasted to hairpin-based silencing designs that generate a largepopulation of siRNA, ranging in size from 20-24 nt that span the lengthof the hairpin (for example, see FIG. 8 ). p19 was tested to determineif it would block the activity of artificial miRNAs or at leastsignificantly reduce it, whereas V2 was tested to determine if it wouldallow silencing when combined with artificial miRNAs.

The influence of V2 and p19 on an example of amiRNA activity was testedin two ways, namely amiRNA silencing of a gene encoding a component ofchlorophyll biogenesis (AtPDS) and amiRNA targeting of a gene encodingan enzyme in seed oil biosynthesis (AtFAD2). Silencing of the geneencoding Phytoene Desaturase (PDS) using RNAi generated a bleached leafphenotype (Helliwell et al., 2002). A pri-amiRNA sequence of 915 bplength targeting AtPDS was chemically synthesised using the mil59b as atemplate pri-amiRNA (Millar and Gubler, 2005) (SEQ ID NO: 52) and clonedinto a 35S expression vector, generating pCW159 (35S-pri-amiRNA-PDS).This 35S-pri-amiRNA-PDS-ocs3′ fragment was removed by enzymaticdigestion and ligated into the binary vectors expressing 35S-p19 and35S-V2, generating expression constructs for pCW160(35S-19+35S-pri-amiRNA-PDS) and pCW161 (35S-V2-ocs+35S-pri-amiRNA-PDS),respectively. These constructs, pCW159, pCW160 and pCW161 wereintroduced into Agrobacterium and used to stably transform Arabidopsisthaliana of the Col-0 ecotype. Seeds of dipped plants were selected onkanamycin selection media and the numbers of bleached and non-bleachedtransformed seedlings were counted.

Seedlings transformed with the control 35S-pri-amiRNA-PDS, therebyhaving only the amiRNA silencing construct in the absence of silencingsuppressor, were almost all bleached and survived in tissue culture foronly about 3 weeks. Seedlings transformed with 35S-V2+35S-pri-amiRNA-PDSalso showed only the bleached phenotype and were indistinguishable fromseedling transformed with 35S-pri-amiRNA-PDS. In contrast, seedlingstransformed with the 35S-p19+35S-pri-amiRNA-PDS remained green andviable. These results indicate that V2 did not interfere with thebiogenesis of amiRNA in a seedling context and allowed the miRNAconstruct to silence the endogenous gene. In contrast, p19 blocked theaction of the miRNA, amiRNA-PDS, presumably by binding the 21 nt dsRNAduplexes generated during the processing of the amiRNA.

As described in the earlier Examples, FAD2 desaturates 18:1-PC to18:2-PC, and ablation of this gene via a hairpin RNA resulted inelevated levels of 18:1. A pri-amiRNA having a length 913 bp, using thesame miRNA195b vector template sequence as for the PDS miRNA construct,was designed to target the AtFAD2 gene (SEQ ID NO: 53) and inserted intoa seed specific expression vector, FP1-pORE4, generating pJP1106. Thisvector was introduced into Agrobacterium and used to transformArabidopsis of the Col-0 ecotype—this ecotype had an active FAD2 geneand consequently low levels of 18:1 in seed oils. Stably transformedplants of this ecotype were isolated and analysed for oleic acid contentin seed oil. One line, HX13, was selected as having greatly increasedlevels of 18:1 in seeds oils, and this event was made homozygous viaself-fertilisation of plants into the T4 generation. Homozygous HX13plants were then super-transformed with Agrobacterium containing binaryvectors expressing either FP1-p19 or FP1-V2 and Ti seeds of these plants(FP1-amiRNA-AtFAD2+FP1-V2) or (Fp1-amiRNA-AtFAD2+FP1-p19) selected andgrown into T2 seed before analysis of the oil profile.

HX13 plants expressing Fp1-amiRNA-AtFAD2 exhibited an oleic acid contentof 65% as a percentage of the total fatty acids in the seedoil. HX13plants co-expressing FP1-V2 in addition to the amiRNA construct wereindistinguishable to those containing the amiRNA construct alone,indicating that V2 did not interfere with amiRNA function in seeds andallowed the miRNA silencing construct to silence as in the absence ofthe silencing suppressor. In contrast, HX13 plants co-expressing theFP1-p19 construct exhibited markedly reduced 18:1 levels in seedoil,dropping to levels similar to those in seeds of the untransformed Col-0ecotype. These results indicated that p19 suppressed amiRNA-basedsilencing of an endogenous gene in seeds.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/580,574 filed 27Dec. 2011, the entire contents of which are incorporated herein byreference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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The invention claimed is:
 1. A plant cell comprising, i) apolynucleotide of interest which encodes a target RNA, ii) a firstexogenous polynucleotide encoding a double stranded RNA (dsRNA) moleculewhich comprises a stem-loop and a first nucleotide sequence which iscomplementary to a region of the target RNA encoded by thepolynucleotide of interest, iii) a second exogenous polynucleotideencoding a silencing suppressor polypeptide, iv) a third exogenouspolynucleotide, different to the first and second exogenouspolynucleotides and the polynucleotide of interest, which encodes an RNAof interest, and v) a reduced level of the target RNA encoded by thepolynucleotide of interest and/or the amount of a protein encoded by thetarget RNA when compared to a corresponding cell lacking the firstexogenous polynucleotide, wherein each exogenous polynucleotide isoperably linked to one or more promoters that are capable of directingexpression of the polynucleotide in the cell, wherein the first andsecond exogenous polynucleotides form part of one DNA construct, andwherein the silencing suppressor polypeptide comprises amino acidshaving the sequence set forth in any one of SEQ ID NOs 39 to 51, or asequence which is at least 95% identical to the sequence set forth inany one of SEQ ID NOs 39 to
 51. 2. The plant cell of claim 1, whereinthe polynucleotide of interest is an endogenous gene of the cell or agene of a pathogen of the cell.
 3. The plant cell of claim 1, whereinthe first and second exogenous polynucleotides form part of one DNAconstruct which is integrated into the genome of the cell.
 4. The plantcell of claim 1, wherein the first, second and third exogenouspolynucleotides form part of one DNA construct which is integrated intothe genome of the cell.
 5. The plant cell of claim 1, wherein at leastthe second exogenous polynucleotide is integrated into the genome of thecell.
 6. The plant cell of claim 1, wherein the cell comprises at leasta 25% reduction in the level of the target RNA encoded by thepolynucleotide of interest and/or amount of protein encoded by thetarget RNA when compared to a corresponding cell lacking the firstexogenous polynucleotide.
 7. The plant cell of claim 1, wherein thedsRNA molecule, or a processed RNA product thereof, comprises at least19 consecutive nucleotides which is at least 95% identical to thecomplement of a region of the target RNA, and wherein the region of thetarget RNA is i) within a 5′ untranslated region of the target RNA, ii)within a 5′ half of the target RNA, iii) within a protein-encodingopen-reading frame of the target RNA, iv) within a 3′ half of the targetRNA, or v) within a 3′ untranslated region of the target RNA.
 8. Theplant cell of claim 1, wherein the dsRNA molecule is a microRNA (miRNA)precursor and/or wherein the processed RNA product thereof is a miRNA.9. The plant cell of claim 1, wherein the third exogenous polynucleotideencodes a protein or microRNA precursor.
 10. The plant cell of claim 1,wherein the cell further comprises at least one, at least two, at leastthree, at least four or at least five additional, different exogenouspolynucleotides each encoding different RNAs of interest.
 11. The plantcell of claim 10, wherein the additional, different exogenouspolynucleotides form part of one DNA construct.
 12. The plant cell ofclaim 1, wherein the cell further comprises at least one, at least two,at least three, at least four or at least five additional, differentexogenous polynucleotides each independently encoding different dsRNAmolecules which comprise different nucleotide sequences which arecomplementary to a region of different target RNAs encoded by differentpolynucleotides of interest, and/or different nucleotide sequences whichare complementary to different regions of the same target RNA.
 13. Theplant cell of claim 12, wherein the additional polynucleotides form partof the same DNA construct.
 14. The plant cell of claim 1, wherein thefirst exogenous polynucleotide encodes more than one miRNA whichindependently comprise different nucleotide sequences which arecomplementary to a region of different target RNAs encoded by differentpolynucleotides of interest, and/or different nucleotide sequences whichare complementary to different regions of the same target RNA.
 15. Theplant cell of claim 1, wherein the exogenous polynucleotides areoperably linked to different promoters.
 16. The plant cell of claim 1,wherein the second exogenous polynucleotide was introduced into the cellon a vector other than a viral vector.
 17. The plant cell of claim 1,wherein the dsRNA molecule is a hairpin RNA.
 18. The plant cell of claim1, wherein the silencing suppressor polypeptide comprises amino acidshaving a sequence which is at least 96% identical to the sequence setforth in any one of SEQ ID NOs 39 to
 51. 19. The plant cell of claim 1,wherein the silencing suppressor polypeptide comprises amino acidshaving the sequence set forth in any one of SEQ ID NOs 39 to
 51. 20. Aprocess for selecting a plant cell with a desired property resultingfrom an increased level of an RNA of interest and/or amount of proteinencoded by the RNA of interest, and a reduced level of target RNAencoded by a first polynucleotide of interest and/or amount of theprotein encoded by the target RNA, the process comprising: i) obtainingone or more plant cells comprising, (a) a polynucleotide of interestwhich encodes a target RNA, b) a first exogenous polynucleotide encodinga double stranded RNA (dsRNA) molecule which comprises a stem-loop and afirst nucleotide sequence which is complementary to a region of thetarget RNA encoded by the polynucleotide of interest, c) a secondexogenous polynucleotide encoding a candidate silencing suppressorpolypeptide, d) a third exogenous polynucleotide, different to the firstand second exogenous polynucleotides and the polynucleotide of interest,which encodes an RNA of interest, and e) a reduced level of the targetRNA encoded by the polynucleotide of interest and/or the amount of aprotein encoded by the target RNA when compared to a corresponding celllacking the first exogenous polynucleotide, wherein each exogenouspolynucleotide is operably linked to one or more promoters that arecapable of directing expression of the polynucleotide in the cell,wherein the first and second exogenous polynucleotides form part of oneDNA construct, and wherein the candidate silencing suppressorpolypeptide comprises amino acids having the sequence set forth in anyone of SEQ ID NOs 39 to 51, or a sequence which is at least 95%identical to the sequence set forth in any one of SEQ ID NOs 39 to 51,ii) analysing the cell(s) for the desired property, iii) if the cell(s)does not have the desired property, substituting the second exogenouspolynucleotide encoding the candidate silencing suppressor polypeptidewith a second exogenous polynucleotide encoding a different candidatesilencing suppressor polypeptide comprising amino acids having thesequence set forth in any one of SEQ ID NOs 1 or 39 to 51, or having asequence which is at least 95% identical to the sequence set forth inany one of SEQ ID NOs 1 or 39 to 51, and analysing the resultant cell(s)for the desired property, iv) if necessary, repeating step iii) untilthe desired property is obtained, and v) selecting a plant cell with thedesired property.